Development of universal cancer drugs and vaccines

ABSTRACT

This invention generally relates to a design and method for developing novel anti-tumor/cancer drugs, vaccines and therapies, using microRNA (miRNA) and its shRNA homologues/derivatives. More particularly, the present invention relates to the use of a nucleic acid composition capable of expressing mir-302-like gene silencing effectors upon delivery into human cells and then silencing mir-302-targeted cell cycle regulators and oncogenes, resulting in an inhibitory effect on tumor/cancer cell growth and metastasis. Mir-302 is the most predominant miRNA found in human embryonic stem (hES) and induced pluripotent stem (iPS) cells, yet its function is unclear. The present invention establishes that in humans mir-302 concurrently suppressed both cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways and eventually blocked over 70% of the G1-S transition. Simultaneously, mir-302 also silences BMI-1, a cancer stem cell marker, and subsequently promotes the tumor suppressor functions of p16Ink4a and p14/p19Arf in inhibiting CDK4/6-mediated cell proliferation. Therefore, the present invention for the first time reveals the tumor suppressor function of mir-302 in humans. This novel finding advances the design and method for developing new cancer drugs, vaccines and therapies directed against multiple kinds of human tumors and cancers, in particular including, but not limited, malignant skin, prostate, breast and liver cancers as well as various tumors.

CLAIM OF THE PRIORITY

The present application claims priority to the U.S. ProvisionalApplication Ser. No. 61/272,169 filed on Aug. 26, 2009, entitled“Mir-302 reprograms somatic cell stemness with high pluripotency in SCNTand the application of chemical reprogramming thereof”. The presentapplication also claims priority to the U.S. Provisional ApplicationSer. No. 61/323,190 filed on Apr. 12, 2010, entitled “Mir-302 inhibitshuman iPS Cell tumorigenecity through co-suppression of CDK2 and CDK4/6pathways”. The present application is a continuation-in-part applicationof the U.S. patent applications Ser. No. 12/149,725 filed on May 7,2008, entitled “Generation of Human Embryonic Stem-Like Cells UsingIntronic RNA”, and the U.S. patent applications Ser. No. 12/318,806filed on Jan. 8, 2009, entitled “Generation of Tumor-Free EmbryonicStem-Like Pluripotent Cells Using Inducible Recombinant RNA Agents”,which are hereby incorporated by reference as if fully set forth herein.

FIELD OF THE INVENTION

This invention generally relates to a design and utilization ofDNA/RNA-based therapeutical drugs and/or vaccines for cancer therapy.More particularly, the present invention relates to the design andmethod of using a nucleic acid composition capable of generating andexpressing small RNA-based gene silencing effectors upon delivery intohuman cells and then silencing mir-302-targeted cell cycle regulatorsand oncogenes, resulting in an inhibitory effect on tumor/cancer cellgrowth and metastasis. Preferably, the small RNA-based gene silencingeffectors include microRNA (miRNA) such as mir-302a, mir-302b, mir-302c,mir-302d, mir-302e, and their precursors (pre-miRNA) as well as manuallyre-designed shRNA/siRNA homologues/derivatives, and a combinationthereof. The human cells of interest include isolated somatic ortumor/cancer cells in vitro, ex vivo and/or in vivo.

BACKGROUND OF THE INVENTION

Mir-302 is the most predominant microRNA (miRNA) found in humanembryonic stem (hES) and induced pluripotent stem (iPS) cells, yet itsfunction is unclear. Previous studies have shown that ectopic expressionof mir-302 is able to reprogram human cancer cells to hES-likepluripotent cells with a distinct slow cell cycle rate and dormantcell-like morphology (Lin et al., 2008). Relative quiescence is adefined characteristic of these mir-302-reprogrammed pluripotent stem(mirPS) cells, while other three/four factors (i.e. Oct4-Sox2-Klf4-c-Mycor Oct4-Sox2-Nanog-Lin28)-induced pluripotent stem (iPS) cells havedramatic proliferative ability and inexorable tumorigenetic tendency(Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007). Despitethe mechanism underlying this anti-proliferative characteristic of mirPScells is largely unknown, we have identified the possible involvement oftwo mir-302-targeted G1-checkpoint regulators, cyclin-dependent kinase 2(CDK2) and cyclin D (Lin et al., 2008). Progression in the eukaryoticcell cycle is driven by activities of cyclin-dependent kinases (CDKs),which forms functional complexes with positive regulatory subunits,cyclins, as well as by negative regulators, CDK inhibitors (CKIs, suchas p14/p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1/Waf1, and p27Kip1).In mammalian cells, different cyclin-CDK complexes are involved inregulating different cell cycle transitions, such as cyclin-D-CDK4/6 forG1 progression, cyclin-E-CDK2 for G1-S transition, cyclin-A-CDK2 forS-phase progression, and cyclinA/B-CDC2 (cyclin-A/B-CDK1) for entry intoM-phase. Thus, it is conceivable that the anti-proliferative function ofmir-302 may result from the co-suppression of CDK2 and cyclin D duringG1-S transition.

However, studies of the mir-291/294/295 family, an analog to humanmir-302 in mouse, revealed a totally different result from the mir-302function in human mirPS cells. In mouse embryonic stem (mES) cells,ectopic expression of mir-291/294/295 promoted fast cell proliferationand G1-S transition through direct silencing of p21Cip1 (also namedCDKN1A) and serine/threonine-protein kinase Lats2 (Wang et al., 2008).This tumor-prone result was presumed due to the tumor suppressor natureof p21Cip1 and Lats2. Transgenic mice lacking p21Cip1/Waf1 were shown todisplay normal development with a defect in the G1 checkpoint control(Deng et al., 1995). Yet, the role of Lats2 remains to be determinedbecause of its function in recruitment of amma-tubulin and spindleformation at the onset of mitosis. Loss of Lats2 in mouse embryos wasfound to cause severe mitotic defects and lethality, indicating thatsilencing of Lats2 may hinder rather than facilitate cell division(Yabuta et al., 2007). Taken together, silencing of p21Cip1 seems to bethe key mechanism underlying such mir-291/294/295-inducedtumorigenecity. Nevertheless, our recent effort to screen the mir-302target site in human p21Cip1 gene shows negative. The same negativeresult was also predicted by online computing programs TARGETSCAN(http://www.targetscan.org/) and PICTAR-VERT(http://pictar.mdc-berlin.de/). Therefore, mir-302 and its analogmir-291/294/295 likely have different functions in hES and mES cells,lending different characteristics to human and mouse iPS cells. Thisfinding suggests that the role of mir-291/294/295 in mES cells cannotserve as an equivalent model for evaluating mir-302 function in hES andiPS cells.

MiRNA is a cytoplasmic inhibitor and often functions to suppress thetranslation of it targeted gene transcripts (mRNAs) with highcomplementarity. The binding stringency between miRNA and its targetgenes determines the real function of a miRNA. Depending on the cellularcondition, miRNA may present different preferences in gene targeting.However, there is no report related to either the concentration effectof mir-302 or the stringency of mir-302-target gene interaction. Toresolve this problem, our present invention provides insight into theseimportant details and for the first time reveals that mir-302 functionsvery differently in human and mouse cells. In humans, mir-302 stronglytargets CDK2, cyclins D1/D2 and BMI-1, but interestingly, not p21Cip1.Unlike mouse p21Cip1, human p21Cip1 does not contain any target site formir-302. This different gene targeting leads to a significant schismbetween respective cell cycle regulations. In mES cells, mir-302silences p21Cip1 and promotes tumor-like cell proliferation (Wang etal., 2008; Judson, 2009), whereas p21Cip1 expression is preserved inhuman mirPS cells and may cause slower cell proliferation and lowertumorigenecity. Additionally, mouse BMI-1 is not a target gene formir-302 either due to lack of a proper target site. We have found thatsilencing of human BMI-1 in human mirPS cells stimulates p16Ink4a/p14ARFexpression to attenuate cell proliferation, whereas mir-302 cannotsilence mouse BMI-1 to raise the same effect in mouse cells. Sincep16Ink4a/p14ARF are elevated while p21Cip1 is not affected in mirPScells, the anti-proliferative function of mir-302 in human cells mostlikely goes through p16Ink4a-Rb and/or p14/19ARF-p53 pathways inaddition to the co-suppression of cyclin-E-CDK2 and cyclin-D-CDK4/6pathways. These distinct targeting preferences of mir-302 to human andmouse genes imply that the mechanisms underlying their cell cycleregulations are fundamentally different in human and mouse cells.

In sum, prior arts overlooked the stringency of miRNA-target geneinteraction and thus misled human mir-302 function into a wrongassumption. To clarify this misunderstanding, our present inventionadopted an inducible mir-302 expression system to reveal a novelfunction of mir-302 in inhibition of human tumor/cancer cell growth, ofwhich our new finding is useful for developing universalanti-tumor/cancer drugs and/or vaccines for cancer therapy as well asprevention. Therefore, there remains a need for effective and safedesigns and methods for utilizing mir-302 and its precursors as well ashomologues/derivatives in drug/vaccine development and cancer therapy.

SUMMARY OF THE INVENTION

The present invention is a design and utilization of DNA/RNA-basedtherapeutical drugs and/or vaccines for cancer therapy. Moreparticularly, the present invention uses a recombinant nucleic acidcomposition capable of expressing small RNA gene silencing effectorsupon delivery into human cells to inhibit the activities ofmir-302-targeted cell cycle regulators and oncogenes, resulting in atumor suppressor effect directed against human tumor/cancer cell growthand metastasis. Preferably, the small RNA gene silencing effectorsinclude microRNA (miRNA) and miRNA-like agents, such as mir-302a,mir-302b, mir-302c, mir-302d, and their hairpin-like microRNA precursors(pre-miRNAs) and manually re-designed small hairpin RNA (shRNA)homologues/derivatives as well as a combination thereof. The designs ofshRNA homologues/derivatives include mismatched and perfectly matchednucleic acid compositions of small hairpin RNA (shRNA) and smallinterfering RNA (siRNA) constructs in a single separate unit or amultiple unit cluster, all of which may improve the target specificityand reduce the copy number of mir-302 required for delivery and therapy.

Native microRNA (miRNA) is sized approximately 18-27 nucleotides (nt) inlength and capable of either directly degrading its targeted messengerRNA (mRNA) or suppressing the translation of its targeted mRNA,depending on their mutual complementarity between miRNA and the targets.The mir-302 family (mir-302s) is a group of highly homologous miRNAsconserved in many mammals. Mir-302s consists of four members which aretranscribed together as a non-coding RNA cluster containing mir-302b,mir-302c mir-302a, mir-302d and mir-367 in a 5′ to 3′ direction (Suh etal., 2004). Recently, the fifth mir-302 member was also found outsidethe familial cluster, namely mir-302e. Although mir-367 and mir-302s areco-expressed but their functions are actually different from each other,we prefer to re-design the mir-302 cluster for expressing only mir-302s.In addition, we also prefer to use manually re-designed hairpin loops,such as 5′-GCTAAGCCAG GC-3′ (SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3′(SEQ.ID.NO.2), to replace the original mir-302 precursor (pre-mir-302)loops for making a more compact cluster for easy delivery andexpression. Normally, mir-302 is only expressed abundantly in mammalianembryonic stem (ES) cells, except mouse ES cells, and quickly decreasedafter cell differentiation and/or proliferation (Tang et al., 2007; Suhet al., 2004). Given that miRNA is characterized as small inhibitoryRNAs capable of silencing its target genes with high complementarity(Bartel, D. P., 2004), mir-302s is likely a key inhibitor responsiblefor preventing errant and premature growth of ES cells during earlyembryogenesis, which may also prevent tumor formation from the stemcells. In fact, ES cells before the morula stage (32-64 cell stage)often present a very slow cell cycle rate. These findings suggest thatmir-302 plays an important role in regulating normal stem cellmaintenance and renewal, which may also help to inhibit tumor/cancerformation.

All mir-302 members share a totally identical sequence in their first5′-seventeen (17) nucleotides, including the entire seed motif5′-UAAGUGCUUC CAUGUUU-3′ (SEQ.ID.NO.3), and contain over 85% homology intheir complete 23-nucleotide mature miRNA sequences. Based on thepredicted results of on-line computing programs TARGETS CAN(http://www.targetscan.org/) and PICTAR-VERT(http://pictar.mdc-berlin.de/), these members currently and concurrentlytarget against almost the same cellular genes, including over 607 humangenes. In addition, mir-302 also shares many overlapping target geneswith mir-93, mir-367, mir-371, mir-372, mir-373, and mir-520 familialmembers, which may provide certain similarity in functionality. Most ofthese target genes are developmental signals and transcriptional factorsinvolved in initiation and/or establishment of lineage-specific celldifferentiation during early embryogenesis (Lin et al., 2008). Many ofthese target genes are also well-known oncogenes. Therefore, thefunction of mir-302s is more likely to suppress the global production ofdevelopmental signals and differentiation-related transcription factorsrather than to create transcriptional stimulation on certain embryonicsignaling pathways like what the previous iPS methods did. Furthermore,since many of these targeted developmental signals anddifferentiation-related transcription factors are oncogenes, mir-302slikely functions as a tumor suppressor to prevent the deviation ofnormal hES cell growth into tumor/cancer formation.

In one preferred embodiment, the inventors have designed and developedan inducible pTet-On-tTS-miR302s expression vector (FIG. 1A) inconjunction with either viral infection, electroporation orliposomal/polysomal transfection methods to deliver mir-302 into normaland/or cancerous human cells. The redesigned mir-302 construct isconsisting of four small non-coding RNA members: mir-302a, b, c and d inone cluster (mir-302s; FIG. 1B). The expression of this mir-302sconstruct is driven by a tetracycline-responsive-element(TRE)-controlled cytomegaloviral (CMV) promoter in response todoxycycline (Dox) stimulation. After infection/transfection, theexpression of mir-302 followed the natural miRNA biogenesis pathway, inwhich the mir-302s construct is co-transcribed with a reporter gene suchas red-shifted fluorescent protein (RGFP), and then further processedinto individual mir-302 members by spliceosomal components and/orcytoplasmic RNaseIII Dicers (FIG. 2A) (Lin et al., 2003). As a result ofthis strategy, miRNA microarray analysis (Example 3) shows that allsense mir-302 members were efficiently expressed in transfected cellsafter Dox stimulation (FIG. 1C). The procedure for transducing themir-302 expression vector into human cells is summarized in FIGS. 2B-C.

By mimicking the natural intronic miRNA biogenesis pathway (FIG. 2A),the inventors have devised an intronic miRNA expression system totranscribe a recombinant RGFP gene, namely SpRNAi-RGFP, which contains aman-made/artificial splicing-competent intron (SpRNAi) capable ofproducing intronic miRNA and/or shRNA-like gene silencing effectors (Linet al., 2003; Lin et al., (2006) Methods Mol. Biol. 342: 295-312). TheSpRNAi is co-transcribed within the pre-mRNA of the SpRNAi-RGFP gene byPol-II RNA polymerases and cleaved out by RNA splicing components.Subsequently, the spliced SpRNAi is further processed into mature genesilencing effectors, such as native miRNAs and man-made shRNAs, so as totrigger specific RNA interference (RNAi) effects on target genes.Meanwhile, after intron splicing, the exons of the SpRNAi-RGFP genetranscript are linked together to form a mature mRNA for translation ofa RGFP reporter protein useful for identifying the miRNA/shRNAexpression. For quantification measurement, one fold RGFP concentrationequaled to four folds the mir-302 concentration. Alternatively, somefunctional protein exons may be used in place of RGFP to provideadditional gene functions, such as hES gene markers for somatic cellreprogramming. Given that there are currently over 1000 native miRNAspecies found in vertebrates without clear function and many more newmiRNAs continue to be identified, our intronic miRNA expression systemmay also serve as a powerful tool for testing these miRNA functions invitro and in vivo.

The SpRNAi intron contains several consensus nucleotide components,consisting of a 5′-splice site, a branch-point (BrP) motif, apoly-pyrimidine tract, and a 3′-splice site. In addition, a hairpinmiRNA or shRNA precursor is inserted in between the 5′-splice site andthe BrP motif. This portion of intron usually forms a lariat structureduring RNA splicing and processing. Moreover, the 3′-end of SpRNAicontains a multiple translational stop codon region (T codon) toincrease the accuracy of intronic RNA splicing and processing. Whenpresented in a cytoplasmic mRNA, this T codon signals the activation ofintracellular nonsense-mediated decay (NMD) system to degrade anyunstructured RNA accumulated in the cell for preventing cytotoxicity.However, the highly structured shRNA and precursor miRNA (pre-miRNA)will be preserved for further Dicer cleavage to form mature siRNA andmiRNA, respectively. For intronic miRNA/shRNA expression, we manuallyincorporate the SpRNAi in the DraII restriction site of a RGFP gene (Linet al., 2006 and 2008). This forms a recombinant SpRNAi-RGFP gene. Thecleavage of RGFP with DraII generates an AG-GN nucleotide break withthree recessing nucleotides in each end, which will form 5′- and3′-splice sites, respectively, after SpRNAi insertion. Because thisintronic insertion disrupts the integrity of RGFP protein, which can berecovered by intron splicing, we are able to determine the expression ofmature miRNA/shRNA through the appearance of red RGFP in the transfectedcells. The RGFP gene also contains multiple exonic splicing enhancers(ESEs) to increase RNA splicing accuracy and efficiency.

In details, the SpRNAi intron contains a 5′-splice site homologous toeither 5′-GTAAGAGK-3′ (SEQ.ID.NO.4) or GU(A/G)AGU motifs (i.e.5′-GTAAGAGGAT-3′, 5′-GTAAGAGT-3′, 5′-GTAGAGT-3′ and 5′-GTAAGT-3′), whileits 3′-end is a 3′-splice site that is homologous to either GWKSCYRCAG(SEQ.ID.NO.5) or CT(A/G)A(C/T)NG motifs (i.e. 5′-GATATCCTGC AG-3′,5′-GGCTGCAG-3′ and 5′-CCACAG-3′). Moreover, a branch point sequence islocated between the 5′- and 3′-splice sites, containing high homology to5′-TACTWAY-3′ (SEQ.ID.NO.6) motifs, such as 5′-TACTAAC-3′ and5′-TACTTAT-3′. The adenosine “A” nucleotide of the branch-point sequencecan form a part of (2′-5′)-linked lariat intron RNA by cellular(2′-5′)-oligoadenylate synthetases and spliceosomes in almost allspliceosomal introns. Furthermore, a poly-pyrimidine tract is closelylocated between the branch-point and 3′-splice site, containing a high Tor C content sequence homologous to either 5′-(TY)m(C/−)(T)nS(C/−)-3′(SEQ.ID.NO.7) or 5′-(TC)nNCTAG(G/−)-3′ (SEQ.ID.NO.8) motifs. The symbolsof “m” and “n” indicate multiple repeats ≧1; most preferably, the mnumber is equal to 1˜3 and the n number is equal to 7˜12. The symbol “−”refers a nucleotide that can be skipped in the sequence. There are alsosome linker nucleotide sequences for the connection of all thesesynthetic intron components. Based on the guideline of 37 CFR 1.822 forsymbols and format to be used for nucleotide and/or amino acid sequencedata, the symbol W refers to an adenine (A) or thymine (T)/uracil (U),the symbol K refers to a guanine (G) or thymine (T)/uracil (U), thesymbol S refers to a cytosine (C) or guanine (G), the symbol Y refers toa cytosine (C) or thymine (T)/uracil (U), the symbol R refers to anadenine (A) or guanine (G), and the symbol N refers to an adenine (A),cytosine (C), guanine (G) or thymine (T)/uracil (U).”

In another preferred embodiment, the present invention is a direct(exonic) mir-302 miRNA/shRNA expression system, which can be used forgenerating mir-302-like gene silencing effectors directly from theexpression system without going through intracellular RNA splicingand/or NMD mechanisms. However, the drawback of this method is that theexpression of mir-302-like gene silencing effectors is not regulated byany intracellular surveillance system, such as NMD, and may thereforeover-saturate the natural miRNA biogenesis pathway to cause cytotoxicity(Grimm et al., 2006). The expression system used for this method can bea linear or circular nucleic acid composition selected from the group ofplasmid, viral vector, lentiviral vector, transposon, retrotransposon,jumping gene, protein-coding gene, non-coding gene, artificiallyrecombinant transgene, and a combination thereof. The mir-302-like genesilencing effectors, including miRNA, shRNA, siRNA and their precursorsas well as homologues/derivatives, are expressed under the control of atissue-specific or non-specific RNA promoter selected from the groupconsisting of type-II RNA polymerase (Pol-II), viral polymerase,type-III RNA polymerase (Pol-III), type-I RNA polymerase (Pol-I), andtetracycline responsive element-controlled RNA polymerase (TRE)promoters. The viral promoters are Pol-II-like RNA promoters isolatedbut not limited from cytomegalovirus (CMV), retrovirus long-terminalregion (LTR), hepatitis B virus (HBV), adenovirus (AMV), andadeno-associated virus (AAV). For example, a lentiviral LTR promoter issufficient to produce up to 5×10⁵ copies of pre-mRNA transcripts percell. It is also feasible to insert a drug-sensitive repressor (i.e.tTS) in front of the RNA polymerase promoter in order to control thetranscription rate of the gene silencing effectors. The repressor can beinhibited by a chemical drug or antibiotics selected from the group ofG418, neomycin, tetracycline, doxycycline, ampicillin, kanamycin,puromycin, and their derivatives, etc.

In another aspect, multiple transgenes and/or vectors expressing variousintronic gene silencing effectors may be used to achieve gene silencingon the mir-302-targeted genes. Alternatively, multiple gene silencingeffectors may be generated from one intronic insert. For example, it hasbeen reported that the ectopic expression of one anti-EGFPpre-miRNA-containing intron in zebrafish generates two different sizemiRNAs, namely miR-EGFP(282/300) and miR-EGFP(280-302), indicating thatone insert of the SpRNAi may generate multiple gene-silencing effectors(Lin et al. (2005) Gene 356: 32-38). In certain cases, intronicgene-silencing effectors can hybridize with a target gene transcript(i.e. mRNA) to form double-stranded siRNAs for triggering secondary RNAinterference (RNAi) effects. Because these gene-silencing effectors areconstantly produced from the transgene vector, it will alleviate theconcerns of fast RNA degradation in vivo. The advantage of this strategyis in its stable delivery through the vector-based transgenetransfection or viral infection, providing a reliable long-term genesilencing efficacy.

Because the stem-loop structures of some native pre-miRNAs are too largeand/or complicated to fit in a miRNA expression system/vector, theinventors often use a manually re-designed tRNA^(met) loop (i.e.5′-(A/U)UCCAAGGGGG-3′), to replace the native pre-miRNA loops. ThetRNA^(met) loop has been shown to efficiently facilitate the export ofmanually re-designed miRNAs from nucleus to cytoplasm through the sameRan-GTP and Exportin-5 transporting mechanisms as native miRNAs do (Linet al., 2005). Advantageously, the present invention now uses a pair ofmanually improved pre-mir-302 loops, including 5′-GCTAAGCCAG GC-3′(SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3′ (SEQ.ID.NO.2), which provide thesame nuclear export efficiency as the native pre-miRNAs but notinterfere with the tRNA exportation. Also, this improvement enhances theformation of mir-302a-mir-302a* and mir-302c-mir-302c* duplexes, whichmay increase the overall function and stability of mir-302s. The designof these new pre-miRNA loops is modified by the combination of thetRNA^(met) loop and the short stem-loops of mir-302b/mir-302a, which arehighly expressed in human ES cells but not in other differentiatedtissue cells. Thus, the use of these recombinant/man-made/artificialhairpin loops in the structure of mir-302 will not interfere with thenative miRNA pathway in human body, resulting in a much lesscytotoxicity and more safety.

The cluster of familial mir-302 pre-miRNAs is formed by hybridizationand linkage/ligation of synthetic mir-302 homologues, consists of fourparts: mir-302a, mir-302b, mir-302c and mir-302d pre-miRNAs in a 5′ to3′ direction (FIG. 1B). All of these manually re-designed mir-302miRNA/shRNA molecules possess an identical 5′-end in their first 17nucleotides [e.g. 5′-UAAGUGCUUC CAUGUUU-3′ (SEQ.ID.NO.3)]. Syntheticoligonucleotides used for DNA recombination of the mir-302 pre-miRNAcluster are listed: including mir-302a-sense, 5′-GTCACGCGTT CCCACCACTTAAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGGATAGATCTCT C-3′ (SEQ.ID.NO.9); mir-302a-antisense, 5′-GAGAGATCTATCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTTTAAGTGGTGG GAACGCGTGA C-3′ (SEQ.ID.NO.10); mir-302b-sense, 5′-ATAGATCTCTCGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCATGTTTTAGTAG GAGTCGCTCA TATGA-3′ (SEQ.ID.NO.11); mir-302b-antisense,5′-TCATATGAGC GACTCCTACT AAAACATGGA AGCACTTACT TTCAAAGTCA CAGAAAGCACTTCCATGTTA AAGTTGAAGG GAGCGAGAGA TCTAT-3′ (SEQ.ID.NO.12);mir-302c-sense, 5′-CCATATGGCT ACCTTTGCTT TAACATGGAG GTACCTGCTGTGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3′(SEQ.ID.NO.13); mir-302c-antisense, 5′-ATGTCTAGAC GCCTCCACTG AAACATGGAAGCACTTACTT CTGTTTCACA CAGCAGGTAC CTCCATGTTA AAGCAAAGGT AGCCATATGG-3′(SEQ.ID.NO.14); mir-302d-sense, 5′-CGTCTAGACA TAACACTCAA ACATGGAAGCACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGCGATCGC AT-3′(SEQ.ID.NO.15); and mir-302d-antisense, 5′-ATGCGATCGC GAACACTCAAACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3′(SEQ.ID.NO.16). Alternatively, we may use the manually re-designed shRNAformed by the hybrid of synthetic miR-302s-sense, 5′-GCAGATCTCGAGGTACCGAC GCGTCCTCTT TACTTTAACA TGGAAATTAA GTGCTTCCAT GTTTGAGTGGTGTGGCGCGA TCGATATCTC TAGAGGATCC ACATC-3′ (SEQ.ID.NO.17) andmir-302s-antisense, 5′-GATGTGGATC CTCTAGAGAT ATCGATCGCG CCACACCACTCAAACATGGA AGCACTTAAT TTCCATGTTA AAGTAAAGAG GACGCGTCGG TACCTCGAGATCTGC-3′ (SEQ.ID.NO.18), in place of the mir-302 pre-miRNA cluster foreasy intronic insertion. The mir-302 shRNA shares over 85% homology toall native mir-302 members and targets the same cellular genes in human.In design of mir-302 homologues, thymine (T) can be used in place ofuracil (U) or vice versa.

For intronic insertion of the mir-302 pre-miRNA/shRNA, given that theinsertion site of the recombinant SpRNAi-RGFP transgene is flanked witha PvuI and an MluI restriction/cloning site at its 5′- and 3′-ends,respectively, the primary insert can be easily removed and replaced byvarious pre-miRNA/shRNA inserts (e.g. mir-302 pre-miRNA/shRNA), whichpossess matched cohesive ends to the PvuI and an MluI restriction sites.By changing the intronic inserts directed against various genetranscripts, the present invention of the intronic mir-302s expressionsystem can be used as a powerful tool for inducing targeted genesilencing in vitro, ex vivo and in vivo. After intronic insertion, themir-302-inserted SpRNAi-RGFP transgene is further inserted into therestriction/cloning site (i.e. a XhoI-HindIII site) of a Dox-induciblepSingle-tTS-shRNA vector to form a pTet-On-tTS-mir302s expression vectorfor intracellular expression (FIG. 1A).

Delivery of the mir-302-expressing nucleic acid composition into humancells can be accomplished using a non-transgenic or transgenic methodselected from the group of liposomal/polysomal/chemical transfection,DNA recombination, electroporation, gene gun penetration,transposon/retrotransposon insertion, jumping gene integration,micro-injection, viral infection, retroviral/lentiviral infection, and acombination thereof. To prevent the risks of random transgene insertionand cell mutation, the inventors preferably use liposomal or polysomaltransfection to deliver the pTet-On-tTS-mir302s vector into the targetedhuman cells (i.e. tumor/cancer cells). The expression of mir-302s isdependent on the activation of the TRE-regulated CMV promoter of thepTet-On-tTS-mir302s vector, in the presence of various Doxconcentrations. Therefore, the present invention provides an induciblemechanism by a defined drug (i.e. Dox) to control the expression ofmir-302s in vitro, ex vivo and/or in vivo, which serves as a secondsafeguard in addition to the intracellular NMD system. As a result ofsuch Dox-mediated control, we did not observe any cytotoxicity of RNAaccumulation or over-saturation in the treated cells. Alternatively, thepresent invention is a constitutive mir-302 expression system capable ofconsistently expressing the mir-302-like gene silencing effectors for acertain period of time. Preferably, the expression of mir-302-like genesilencing effectors is driven by a CMV promoter, which is often silencedafter about one-month activation in human cells due to DNA methylation.Such a one-month activation mechanism is beneficial for cancer therapyto prevent RNA accumulation or over-saturation in the treated cells.

In sum, the present invention has adopted a novel design and strategyfor either inducible or constitutive expression of mir-302-like genesilencing effectors in the transfected cells. Mir-302-like genesilencing effectors include mir-302a, mir-302b, mir-302c, mir-302d, andtheir hairpin-like microRNA precursors (pre-miRNAs) as well as manuallyre-designed small hairpin RNA (shRNA) homologues/derivatives, and acombination thereof. In one preferred embodiment, the present inventionprovides a design and method for using a recombinant nucleic acidcomposition capable of being delivered, transcribed and processed intomir-302-like gene silencing effectors in targeted human cells and thusinducing specific gene silencing effects on mir-302-targeted cell cycleregulators and oncogenes in the cells, comprising the steps of: a)providing a recombinant nucleic acid composition capable of beingdelivered, transcribed and processed into at least a gene silencingeffector interfering a plurality of cellular genes targeted by mir-302,and b) treating a cell substrate with said recombinant nucleic acidcomposition. The transcription of mir-302-like gene silencing effectorsis driven either by a constitutive (i.e. CMV) or drug-inducible (i.e.TRE-CMV) promoter. Preferably, the drug-inducible recombinant nucleicacid composition is a Tet-On vector containing a recombinant transgeneinserted with either a recombinant mir-302 family cluster (mir-302s;hybrid of SEQ.ID.NOs.9-16) or a manually re-designed mir-302 shRNAhomologue (i.e. hybrid of SEQ.ID.NOs.17 and 18). The cell substrate mayexpress the mir-302 target genes either in vitro, ex vivo or in vivo. Bysilencing the mir-302-targeted cell cycle regulators and oncogenes, thepresent invention is able to suppress cell tumorigenecity and reprogramthe treated cells into non-tumor/cancer cells.

Using the present invention, the inventors have gathered evidence forthe success of mir-302-mediated tumor/cancer therapy in six areas:First, transfection of mir-302 in normal human cells (mirPS-hHFC) causescell cycle attenuation but not apoptosis nor cell death (FIGS. 1D-F andFIGS. 3A-C). Second, transfection of mir-302 in human normal cells isable to reprogram the cells into a stem cell-like state, which isbeneficial for healing damaged tissues (FIGS. 4A-B and FIGS. 5A-D).Third, transfection of mir-302 in human tumor/cancer cells (mirPS-MCF7,mirPS-HepG2 and mirPS-NTera2) strongly inhibits tumor/cancer celltumorigenecity and causes >98% cell death or apoptosis (FIGS. 6A-E).Fourth, mir-302 can inhibit tumor/cancer cell tumorigenecity not onlythrough co-suppression of multiple cell cycle regulators, such as CDK2,cyclin D1/D2 and BMI-1, but also activation of tumor suppressors,p16INK4a and p14/p19Arf (FIGS. 7A-D). Fifth, In vivo delivery of mir-302into tumors can inhibit >90% tumor cell growth (FIGS. 8A-C). Last,mir-302 does not cause cell senescence through telomere shortening(FIGS. 9A-C). Furthermore, the inventors have successfully usedpolysome/liposome-based transfection to deliver the mir-302-like genesilencing effectors into targeted tumor cells in vivo, preventing therisks of retroviral infection and transgenic mutation (FIGS. 8A-C).These findings provide strong evidence for using mir-302-like genesilencing effectors as therapeutic drugs and/or vaccines fortumor/cancer therapy. Given that mir-302 also functions to rejuvenatehuman cell sternness (Lin et al., 2008), the present invention mayprovide beneficial applications in both stem cell and cancer therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring particularly to the drawings for the purpose of illustrationonly and not limitation, there is illustrated:

FIGS. 1A-F show inducible mir-302 expression and its effect on normalhuman hair follicle cell (hHFC) proliferation. (A) Construct of theDox-inducible pTet-On-tTS-miR302s vector. (B) Structure of the mir-302familial cluster (mir-302s). (C) MiRNA microarray analysis of inducedmir-302 expression at 6 hours after 10 μM Dox treatment (n=3, p<0.01).(D) Northern and western blot analyses of the dose-dependent mir-302effect on the expression patterns of core reprogramming factorsOct3/4-Sox2-Nanog and melanocytic marker genes TRP1 and cytokeratin 16(n=5, p<0.01). (E) Bar charts of flow cytometry analyses showing thedose-dependent mir-302 effect on the changes of mitotic (M phase) anddormant (G0/G1 phase) mirPS-hHFC populations. (F) Mir-302-inducedapoptotic DNA laddering effects after treatment of 10 μM Dox in variousmirPS cell lines.

FIGS. 2A-C depict the biogenesis of mir-302s and generation of mirPScells. (A) Mechanism of intronic mir-302 biogenesis. The mir-302familial cluster was transcribed with a gene encoded for red fluorescentprotein (RGFP) and then further spliced into individual mir-302 membersby spliceosomal components and cytoplasmic RNaseIII Dicers, while theRGFP served as a indicator for mir-302 production. One fold RGFPconcentration equaled to four folds the mir-302 concentration. (B)Schematic procedure for mir-302 transfection withliposome/polysome/electroporation. The inducible mir-302-expressingpTet-On-tTS-miR302s vector (FIG. 1A) was transduced into adult hHFC byelectroporation at 300-400 volts for 150 μsec in a hypo-osmolar PHbuffer (200 μl; Eppendorf). In each test, 10 μg of thepTet-On-tTS-miR302s vector was used to transfect 200,000 cultured hHFCderived from as few as two human hair follicles (dermal papillae). Afterdoxycycline (Dox)-induced expression, the biogenesis of mir-302 reliedon the natural intronic miRNA pathway.

FIGS. 3A-C show the changes of mirPS-hHFC cell properties by Dox-inducedmir-302 expression (Dox=5 or 10 μM). (A) Changes of cell morphology andcell cycle rate before and after Dox-induced reprogramming. Each cellDNA content respective to cell cycle stages was shown by a chart of flowcytometry analysis above the cell morphology (n=3, p<0.01). The first(left) and second (right) peaks of the charts represented the ratios ofresting G0/G1 and mitotic M phase cells in the entire tested cellpopulation, respectively. Scale bars=100 μm. (B) Time-course formationof embryoid body (EB) from a single mirPS-hHFC cell after limitingdilution. The cell cycle was estimated to be approximately 20-24 hoursat start but gradually accelerated after 72 hours. Scale bars=100 μm.

FIGS. 4A-B show analysis of pluripotent marker expression and in vivopluripotent differentiation/assimilation. (A) Northern blot analysis ofhES marker gene expression patterns induced by a high mir-302concentration in mirPS cells compared to those in hES WA01-H1 (H1) andWA09-H9 (H9) cells (n=5, p<0.01). After treatment of 7.5 μM Dox, mir-302concentration was raised to over 30% higher than that of H1 and H9 cells(>30 fold higher than untreated hHFCs) and began inducing co-expressionof the major pluripotent markers Oct3/4, Sox2, Nanog, Lin28 andundifferentiated embryonic cell transcription factor 1 (UTF1). (B)Assimilation of mirPS cell-differentiated tissues into the surroundingtissues around the injection sites of immunocompromised SCID-beige miceone week after the implantation. White arrows indicated the direction ofinjection. Intercalated disks were marked by yellow triangles. ThemirPS-hHFC-derived tissue cysts were not grown in mouse organs/tissuesother than uterus and peritoneal cavity. In view of this, we furtherdissected and examined the surrounding tissue formation around theinjection sites one week after the implantation. One day prior todissection, the mice were treated with 10 μg Dox by tail vein injection.We observed that the RGFP-positive mirPS cells differentiated into thesame cell types as the surrounding tissues in the implanted sites,including gut epithelium after intraperitoneal injection, cardiac muscleafter heart puncture, and skeleton muscle by dorsal flank injection. Notonly that, the assimilated mirPS cells also expressed the sametissue-specific makers as the surrounding tissues, such as MUC2 for gutepithelium, troponin T type 2 (cTnT) for cardiac muscle, and myosinheavy chain (MHC) for skeleton muscle.

FIGS. 5A-D show gain of hES-like properties in Dox-induced mirPS-hHFCs(Dox=10 μM). (A) Analysis of global gene expression patterns before andafter mir-302-induced somatic cell reprogramming (SCR), using humangenome GeneChip U133 plus 2.0 arrays (Affymetrix; n=3, p<0.01-0.05). (B)HpaII cleavage showing the vast loss of global CpG methylation,identified by increased presence of smaller DNA fragments, at agenome-wide scale in mirPS cells treated with 10 μM but not 5 μM Dox.(C) Bisulfite DNA sequencing in the promoter regions of Oct3/4 andNanog, showing the detailed methylation maps. Black and white circlesindicate the methylated and unmethylated cytosine sites, respectively.(D) Pluripotent differentiation of mirPS-hHFCs into teratoma-like tissuecysts, containing various tissues derived from all three embryonic germlayers.

FIGS. 6A-E show in vitro tumorigenecity assays of varioustumor/cancer-derived mirPS cells in response to mir-302 expressioninduced by 10 μM Dox. (A) Changes of cell morphology and cell cycle ratebefore and after Dox-induced mir-302 expression. Each cell DNA contentrespective to cell cycle stages was shown by a chart of flow cytometryanalysis above the cell morphology (n=3, p<0.01). (B) Bar charts of flowcytometry analyses showing the dose-dependent mir-302 effect on thechanges of mitotic (M phase) and dormant (G0/G1 phase) cell populationsof various tumor/cancer-derived mirPS cells. (C) Functional analysis ofmir-302-suppressed tumor invasion in Matrigel chambers (n=4, p<0.05).(D) Comparison of cell adhesion to the hBMEC monolayer before and afterDox-induced mir-302 expression (n=4, p<0.05).

FIGS. 7A-D show luciferase 3′-UTR reporter assays of mir-302-inducedgene silencing effects on targeted G1-checkpoint regulators. (A)Constructs of the luciferase 3′-UTR reporter genes, which carry eithertwo normal (T1+T2) or two mutant (M1+M2), or a mixture of both (T1+M2 orM1+T2), mir-302 target sites in the 3′-UTR. The mutant sites contained amismatched TCC motif in place of the uniform 3′-CTT end of the normaltarget sites. (B) Effects of Dox-induced mir-302 on the luciferaseexpression (n=5, p<0.01). Dox=5 or 10 μM. CCND1 and CCND2 refer tocyclin D1 and D2, respectively. (C) and (D) Western blot analysesshowing the changes of major mir-302-targeted G1-checkpoint regulatorinduced by high (10 μM Dox) and low (5 μM Dox) mir-302 concentrations inmirPS cells compared to those found in hES H1 and H9 cells (n=4,p<0.01).

FIGS. 8A-C show In vivo tumorigenecity assays of mirPS-NTera2 cells inresponse to either constitutive mir-302s (NTera2+mir-302s) or mir-302d*(NTera2+mir-302d*) expression (n=3, p<0.05). Mir-302s and mir-302d* weretranscribed from the pCMV-miR302s and pCMV-miR302d* vectors in thetransfected neoplastic Tera-2 (NTera2) cells, respectively. (A)Morphological evaluation of average tumor sizes three weeks after thein-situ injection (post-is). All tumors were localized in the originalimplant sites (black arrows). No signs of cachexia or tumor metastasiswere observed in all tested mice. (B) Northern and western blot analysesand (C) Immunohistochemical staining analyses of the in vivo mir-302effect on the expression patterns of core reprogramming factorsOct3/4-Sox2-Nanog and mir-302-targeted G1-checkpoint regulators CDK2,cyclins D1/D2 and BMI-1 as well as p16Ink4a and p14Arf.

FIGS. 9A-C show analyses of telomerase activity in mirPS-hHFC andvarious tumor/cancer-derived mirPS cell lines in response to the mir-302expression induced by 10 μM Dox. (A) Telomerase activities shown by TRAPassays (n=5, p<0.01). Telomerase activity was sensitive to RNasetreatment (hHFC+RNase). (B) Western blotting confirming the consistentincrease of hTERT and decrease of AOF2 and HDAC2 expression in variousmirPS cell lines (n=5, p<0.01). (C) Telomerase activities measured bytelomerase PCR ELISA assays (OD470-OD680; n=3, p<0.01).

FIGS. 10A-D show analyses of mir-302-induced silencing effects on itstargeted epigenetic genes. (A) Constructs of the luciferase 3′-UTRreporter genes, which carry either two normal (T1+T2) or two mutant(M1+M2), or a mixture of both (T1+M2 or M1+T2), mir-302 target sites inthe 3′-UTR. The mutant sites contained a mismatched TCC motif in placeof the uniform 3′-CTT end of the normal target sites. (B) Effects ofDox-induced mir-302 on the luciferase expression (n=5, p<0.01). (C) and(D) Western blot analyses showing the changes of major mir-302-targetedepigenetic gene expression induced by high (10 μM Dox) and low (5 μMDox) mir-302 concentrations in mirPS cells compared to those found inhES H1 and H9 cells (n=4, p<0.01).

FIG. 11 depicts the proposed mechanism of mir-302-mediated SCR and cellcycle regulation. Based on our previous and current studies, twocollateral events were discovered. First, reprogramming is initiated bystrong silencing of multiple epigenetic regulators AOF1/2, MECP1/2 andHDAC2, leading to global genomic DNA demethylation, wherebyre-activating hES cell marker genes essential for SCR induction (markedin gray). Second, cell cycle attenuation is caused by co-suppression ofG1-checkpoint regulators CDK2, cyclins D1/D2 and BMI-1 as well asactivation of p16Ink4a and p14/p19Arf to quench all cellular activitiesready for SCR (marked in black). Quiescence at this dormant G0/G1 statealso prevents the possible random growth and/or tumor-liketransformation of the reprogrammed pluripotent stem cells. Collectively,the synergistic effect of these two events results in a more accurateand safe reprogramming process, by which pre-mature cell differentiationand tumorigenecity are both inhibited.

DETAILED DESCRIPTION OF THE INVENTION

Although specific embodiments of the present invention will now bedescribed with reference to the drawings, it should be understood thatsuch embodiments are by way of example only and merely illustrative ofbut a small number of the many possible specific embodiments which canrepresent applications of the principles of the present invention.Various changes and modifications obvious to one skilled in the art towhich the present invention pertains are deemed to be within the spirit,scope and contemplation of the present invention as further defined inthe appended claims.

The present invention provides a novel nucleic acid composition andmethod for inhibiting the proliferation and tumorigenecity of humantumor/cancer cells, using recombinant mir-302-like gene silencingeffectors. Unlike previous shRNA designs, the presently invented shRNAsmay contain a mismatched stem-arm similar to the precursors of nativemir-302 (pre-mir-302). Further, the presently invented shRNAs may alsocontain an improved pre-mir-302 stem-loop, such as 5′-GCTAAGCCAG GC-3′(SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3′ (SEQ.ID.NO.2), which can providethe same nuclear export efficiency as native pre-miRNAs but notinterfere with the tRNA exportation. Without being bound by anyparticular theory, such an anti-proliferative and anti-tumorigeneticeffect of the present invention is directed to a newly discoveredmir-302-mediated gene silencing mechanism, triggered by transfection ofa recombinant nucleic acid composition capable of expressing either amir-302 family cluster (mir-302s) or a mir-302-homologous shRNA. All ofthe manually re-designed mir-302 miRNA/shRNA molecules possess anidentical 5′-end in their first 17 nucleotides, 5′-UAAGUGCUUC CAUGUUU-3′(SEQ.ID.NO.3). The protocols for constructing the mir-302-like genesilencing effectors and the nucleic acid composition expressing mir-302are described in Examples 2 and 3. In design of sequences homologous tomir-302, thymine (T) can be used in place of uracil (U).

To address the mechanistic role of mir-302 in human cell cycle, wedesigned an inducible pTet-On-tTS-miR302s expression vector (FIG. 1A;Example 2) to transfect normal and cancerous human cells. Mir-302 is ahES-specific microRNA (miRNA) family that consists of four smallnon-coding RNA members, mir-302b, c, a, and d, in one cluster (mir-302s;FIG. 1B) (Suh et al., 2004). In our design, the expression of mir-302swas driven by a tetracycline-response-element (TRE)-controlledcytomegaloviral (CMV) promoter in response to doxycycline (Dox)stimulation. After transfection, the biogenesis of mir-302 followed thenatural intronic miRNA pathway, in which mir-302s was transcribed with areporter gene encoded for red fluorescent protein (RGFP) and thenfurther spliced into individual mir-302 members by spliceosomalcomponents and cytoplasmic RNaseIII Dicers (FIG. 2A) (Lin et al., 2008).For quantification measurement, one fold RGFP concentration equaled tofour folds the mir-302 concentration. MiRNA microarray analysisconfirmed that all mir-302 members except mir-302b* were efficientlyexpressed in transfected cells after Dox stimulation (FIG. 1C; Example3). The procedure for transfecting cells with the pTet-On-tTS-miR302sexpression vector is summarized in FIGS. 2B-C.

Moreover, the mir-302-expressing nucleic acid composition, such aspTet-On-tTS-miR302s, may contain a Kozak consensus translationinitiation site to increase translation efficiency in eukaryotic cells,multiple SV40 polyadenylation signals downstream of themir-302-expressing construct, a pUC origin of replication forpropagation in prokaryotic cells, at least two restriction sites forincorporation of the mir-302-expressing construct (i.e. SpRNAi-RGFP)into the nucleic acid composition, an optional SV40 origin forreplication in mammalian cells that express the SV40 T antigen, and anoptional SV40 early promoter for expressing an antibiotic resistancegene in replication-competent prokaryotic cells. The expression ofantibiotic resistance genes is used to serve as a selective marker forisolating positive clones with the transgene expression. The antibioticsare selected from the group consisted of G418, neomycin, puromycin,penicillin G, ampicillin, kanamycin, streptomycin, erythromycin,spectromycin, phophomycin, tetracycline, doxycycline, rifapicin,amphotericin B, gentamycin, chloramphenicol, cephalothin, tylosin, and acombination thereof.

Mir-302 Attenuates the Normal Cell Cycle Rate without Causing Apoptosis

Our previous studies have shown that increasing mir-302 expression inhuman melanoma Colo-829 and prostate cancer PC3 cells reprogrammed thesemalignant cancer cells into a hES-like pluripotent state (Lin et al.,2008). During this somatic cell reprogramming (SCR) process, mir-302caused apoptosis in >98% of the cancer cell population and greatlyreduced the proliferation rate of the remaining (<2%) reprogrammedcells. Although this feature may benefit cancer therapy, it is uncertainhow mir-302 functions in normal human cells. To evaluate this effect, weintroduced the inducible pTet-On-tTS-miR302s expression vector intonormal human hair follicle cells (hHFCs). hHFCs were chosen due to theirabundance, accessibility and fast growth. Following an increase of Doxconcentration up to 10 μM, we observed that the core reprogrammingfactors Oct4-Sox2-Nanog were concurrently stimulated by a threshold ofDox>7.5 μM (FIG. 1D; Example 5) and meanwhile the proliferative cellpopulation was reduced by 70% from 37%±2% to 11%±2% (FIG. 1E, M phase;Example 7). Accordingly, the dormant cell population was increased by41% from 56%±3% to 79%±5% (FIG. 1E, G0/G1 phase; Example 7), reflectinga strong anti-proliferative effect similar to our previous observationin mir-302-reprogrammed pluripotent stem cells (mirPS cells; Lin et al.,2008). However, the mir-302-reprogrammed hHFC cells (mirPS-hHFC) did notdisplay any detectable sign of apoptotic DNA laddering or cell death(FIG. 1F; Example 6), indicating that normal cells are more tolerablethan tumor/cancer cells to the anti-proliferative effect of mir-302. Itis conceivable that tumor/cancer cells are very difficult to survive insuch a dormant state due to their high metabolism and rapid growthexpansion.

Most notably, when treated with Dox>7.5 μM, mirPS-hHFC morphology waschanged from a spindle to a sphere shape, resembling a dormant cell(FIGS. 3A-B, red RGFP-positive cells). The cellular mir-302concentration stimulated by 7.5 μM Dox was approximately 1.3 folds thelevel in the hES H1 and H9 cells (FIG. 4A; Example 4). At this higherlevel, mirPS-hHFCs strongly expressed Oct3/4, Sox2, Nanog, and otherstandard hES cell markers (FIG. 4A). Microarray analysis of global geneexpression further showed that approximately half of the transcriptomechanged from a somatic hHFC mode to a uniform hES-like expressionpattern sharing an average of >93% similarity to that of H1/H9 cells(FIG. 5A; Example 8). Global genomic DNA demethylation, the first signof SCR initiation, was also clearly detected in these mirPS-hHFCsidentical to those demethylation patterns in H1/H9 cells (FIGS. 5B and5C; Example 9). Moreover, each individual mirPS-hHFC cell could growinto a single embryoid body-like colony and the cell division rate was20-24 hours per cycle consistent with the anti-proliferative effect ofmir-302 (FIG. 3C). We particularly noted that these mirPS-hHFCs werepluripotent but not tumorigenetic because they formed teratoma-liketissue cysts only in the uteruses and peritoneal cavities ofpseudopregnant immunocompromised SCID-beige mice. These teratoma-likecysts contained various tissues derived from all three embryonic germlayers, ectoderm, mesoderm and definitive endoderm (FIG. 5D; Example10). Alternatively when xenografted into normal male mice, thesemirPS-hHFCs were assimilated by the surrounding tissues and presentedthe same tissue makers, demonstrating a possible use for healing damagedtissues (FIG. 4B). Taken together, these findings suggest that mir-302can reprogram somatic hHFCs to hES-like iPS cells. Given that Oct3/4 andSox2 are crucial transcription factors for mir-302 expression (Marson etal., 2008; Card et al., 2008), mir-302 may be used in place ofOct3/4-Sox2 for inducing SCR.

Mir-302-induced SCR and cell cycle attenuation are two collateralevents, depending on the mir-302 concentration. We noted that bothevents occur almost simultaneously at a mir-302 concentration over 1.3folds the level in the hES H1/H9 cells, indicating that this specificconcentration is the minimal threshold for initiating both events.Previous studies using a single mir-302 member or at a lowerconcentration equal to the level in H1/H9 cells failed to elicit theseevents. Also, we have shown that the mir-302 concentration induced by alower 5 μM Dox cannot silence either the target sites of the reportergene or the targeted G1-checkpoint regulators. Compared to the naturaldevelopment, early embryonic cells before the morula stage (32-64 cellstage) often present a very slow cell cycle rate similar to that ofmirPS cells. However, such cell cycle regulation is not found inblastocyst-derived hES cells. It is presumable that a lower mir-302concentration in hES cytoplasm may fail to silence the targetedG1-checkpoint regulators and oncogenes. This may also explain whyblastocyst-derived hES cells have dramatic proliferative ability andtend to form tumors. Thus, the present invention may be applied toreduce the tumorigenecity of hES cells for stem cell therapy.

Mir-302 Inhibits Tumorigenecity and Causes Apoptosis in VariousTumor/Cancer Cells

In view of mir-302 function in causing cancer cell apoptosis and cellcycle attenuation, we then investigated the possibility of using mir-302as a universal drug to treat human tumor/cancer cells. As our previousstudies have shown the feasibility of this approach in melanoma andprostate cancer cells (Lin et al., 2008), we further tested human breastcancer MCF7, hepatocellular carcinoma Hep G2, and embryonalteratocarcinoma Tera-2 (NTera-2) cells in the present invention. Asshown in FIGS. 6A-B, all three kinds of tumor/cancer cells werereprogrammed to dormant mirPS cells and formed embryoid body-likecolonies after transfected with the pTet-On-tTS-miR302s vectorstimulated by 10 μM Dox. Mir-302 at this level also induced significantapoptosis (>95%) in all three tumor/cancer cell types (FIG. 1F; Example6). Flow cytometry analysis comparing DNA content to cell cycle stages,further showed a significant reduction in all of the mirPS mitotic cellpopulations (FIG. 6C; Example 7). The mitotic cell population (M phase)was decreased by 78% from 49%±3% to 11%±2% in mirPS-MCF₇, by 63% from46%±4% to 17%±2% in mirPS-HepG2, and by 62% from 50%±6% to 19%±4% inmirPS-NTera2 cells, whereas the resting/dormant cell population (G0/G1phase) was increased by 80% from 41%±4% to 74%±5% in mirPS-MCF7, by 65%from 43%±3% to 71%±4% in mirPS-HepG2, and by 72% from 40%±7% to 69%±8%in mirPS-NTera2 cells, respectively. These results indicate that mir-302can effectively attenuate the fast cell cycle rates and causesignificant apoptosis in these tumor/cancer cells.

In vitro tumorigenecity assays, using Matrigel chambers (cell invasionassay) and cell adhesion to the human bone marrow endothelial cell(hBMEC) monolayer (cell adhesion assay), revealed two moreanti-tumorigenetic effects of mir-302 in addition to itsanti-proliferative feature. Cell invasion assay showed that all threedormant mirPS-tumor/cancer cells lost their ability to migrate (reducedto <1%) while the original tumor/cancer cells aggressively invaded intothe chambered areas supplemented with higher nutrients, representingover 9%±3% of MCF7, 16%±4% of Hep G2 and 3%±2% of NTera-2 cellpopulations (FIG. 6D; Example 11). Consistently, cell adhesion assayalso showed that none of these mirPS-tumor/cancer cells could adhere tohBMECs whereas a significant population of original MCF7 (7%±3%) and HepG2 (20%±2%) cells quickly metastasize into the hBMEC monolayer after 50min incubation (FIG. 6E; Example 12). In sum, all of the findings thusfar strongly and repeatedly suggest that mir-302 is a human tumorsuppressor capable of attenuating fast cell growth, causing tumor/cancercell apoptosis, and inhibiting tumor/cancer cell invasion as well asmetastasis. Most importantly, this novel mir-302 function may offer auniversal treatment against multiple kinds of human cancers/tumors,including but not limited in malignant skin (Colo-829), prostate (PC-3),breast (MCF7) and liver (HepG2) cancers as well as various tumors inview of the variety of different tissue types in teratomas (NTera-2).

Mir-302-Mediated Anti-Proliferation Functions Through Co-Suppression ofCDK2, Cyclins-D1/D2 and BMI-1

To validate the physical interactions between mir-302 and its targetedG1-checkpoint regulators, we used a luciferase 3′-untranslated region(3′-UTR) reporter assay (FIG. 7A; Example 15), which showed thattreatments with various mir-302 concentrations resulted in verydifferent inhibitory effects on the targeted G1-checkpoint regulators,including CDK2, cyclins-D1/D2 and BMI1 polycomb ring finger oncogene(BMI-1). In the presence of 10 μM Dox, mir-302 effectively bound to thetarget sites of CDK2, cyclins D1/D2 and BMI-1 transcripts andsuccessfully silenced >80% of the reporter luciferase expression in alltargets (FIG. 7B; Example 15). Suppression of the real target genes inmirPS cells was also confirmed by western blot analyses consistent withthe results of the luciferase 3′-UTR reporter assay (FIG. 7C; Example5). In contrast, a lower mir-302 expression induced by 5 μM Dox failedto trigger any significant silencing effect on either the target sitesof the reporter gene or the targeted G1-checkpoint regulators, exceptcyclin D2 (FIGS. 7B and 7D), indicating that mir-302 fine-tunes the cellcycle rate in a dose-dependent manner. Given that the G1-S transition ofmammalian cell cycle is normally controlled by two compensatorycyclin-CDK complexes, cyclin-D-CDK4/6 and cyclin-E-CDK2 (Berthet et al.,2006), we found that high concentrated mir-302 inactivated bothcomplexes through simultaneous suppression of CDK2 and cyclins D1/D2,thus blocking both G1-S transition pathways and attenuating the cellcycle rate of the reprogrammed mirPS cells. In hHFCs and mirPS cells,cyclin D3 was expressed at a limited level insufficient to compensatethe loss of cyclins D1/D2 in the mirPS cells.

Accompanying BMI-1 silencing, we further detected a mild increase ofp16Ink4a and p14Arf expression (gain 63%±17% and 57%±13% of the levelsin hHFCs, respectively), whereas no change was found in p21Cip1expression (FIG. 7C). Deficiency of BMI-1, an oncogenic cancer stem cellmarker, has been shown to inhibit G1-S transition through enhancement ofp16Ink4a and p14Arf tumor suppressor activities (Jacobs et al., 1999).In this scenario, 16Ink4a directly inhibits cyclin-D-dependent CDK4/6activity in the phosphorylation of retinoblastoma protein Rb and thusprevents Rb from releasing E2F-dependent transcription required for Sphase entry (Parry et al., 1995; Quelle et al., 1995). In addition,p14Arf prevents HDM2 from binding to p53 and permits the p53-dependenttranscription responsible for G1 arrest or apoptosis (Kamijo et al.,1997). However, because embryonic stem cells are known to exempt fromcyclin-D-dependent CDK regulation (Burdon et al., 2002; Jirmanova etal., 2002; Stead et al., 2002), current understanding of cell cycleregulation in hES cells has implicated CDK2 as the main determinant ofthe G1-S transition. As a result, the silencing of CDK2 likelycontributes most of the G1-arrest efficacy in mirPS cells, while theco-suppression of cyclin-D and BMI-1 as well as co-activation ofp16Ink4a and p14Arf may provide additional inhibition againsttumorigenetic signal-induced cell proliferation. Furthermore, the lossof cyclin-D and activation of p16Ink4a may also explain the deficiencyof cyclin-D-dependent CDK activity in embryonic stem cells.

Therefore, the stringency of miRNA-target gene interaction determinesthe real function of the miRNA. Depending on the cellular condition,miRNA may present different preferences in gene targeting. To this, thepresent invention provides insight into these important details and forthe first time reveals that mir-302 functions very differently in humanand mouse cells. In humans, mir-302 strongly targets CDK2, cyclins D1/D2and BMI-1, but interestingly, not p21Cip1. Unlike mouse p21Cip1, humanp21Cip1 does not contain any target site for mir-302. This differentgene targeting leads to a significant schism between respective cellcycle regulations. In mES cells, mir-302 silences p21Cip1 and promotestumor-like cell proliferation (Wang et al., 2008; Judson, 2009), whereasp21Cip1 expression is preserved in human mirPS cells and may causeslower cell proliferation and lower tumorigenecity. Additionally, mouseBMI-1 is not a target gene for mir-302 either due to lack of a propertarget site. We have shown that silencing of human BMI-1 in mirPS cellsslightly stimulates p16Ink4a/p14ARF expression to attenuate cellproliferation, whereas mir-302 cannot silence mouse BMI-1 to raise thesame effect. Since p16Ink4a/p14ARF were elevated while p21Cip1 was notaffected in mirPS cells, the anti-proliferative and anti-tumorigeneticeffects of mir-302 in human cells most likely goes through p16Ink4a-Rband/or p14/19ARF-p53 pathways in addition to the co-suppression ofcyclin-E-CDK2 and cyclin-D-CDK4/6 pathways. These distinct targetingpreferences of mir-302 to human and mouse genes imply that themechanisms underlying their cell cycle regulations are fundamentallydifferent.

Treatment of Mir-302 Eliminates >90% of Tumor Cell Growth In Vivowithout Changing Stem Cell Pluripotency

After identifying the tumor suppressor function of mir-302 and itsdifferent effects between normal and tumor/cancer cells, we tested thepossible use of mir-302 as a drug for treating NTera2-derived teratomasin eight-week-old male athymic mice (BALB/c nu/nu strain) (Example 13).The neoplastic Tera-2 (NTera-2) cell line is a pluripotent humanembryonal teratocarcinoma cell line that can differentiate into avariety of primitive somatic tissues in vivo, in particular primitiveglandular and neural tissues (Andrews et al., 1984). Due to itspluripotency, NTera2-derived teratoma may serve as a model for treatingvarious tumor types in vivo. For drug delivery, we adopted in situinjection of polyethylenimine (PEI)-formulated pCMV-miR302s expressionvector in close proximity to the tumor site. The pCMV-miR302s vector wasformed by changing the TRE-controlled CMV promoter to a regular CMVpromoter (Example 2), of which the expression duration was approximatelyone month in human cells due to DNA methylation. By injecting up to 10μg of the pCMV-miR302s vector per g mouse weight (the maximal amount ofone shot injection in a mouse), we observed no signs of sickness orcachexia in the mice, indicating the safety of this approach.Histological examination also showed no detectable tissue lesions inbrain, heart, lung, liver, kidney and spleen.

We detected a significant inhibitory effect on teratoma growth afterfive treatments (three-day intervals for each treatment) of 2 μgpCMV-miR302s vector (total 10 μg) per g mouse weight. As shown in FIG.8A (Example 13), when treated with the pCMV-miR302s vector, the averagesize of NTera2-derived teratomas decreased by >89% (11±5 mm³, n=6)compared to that of non-treated ones (104±23 mm³, n=4). In contrast,treating the same amount of PEI-formulated antisense-mir-302d expressionvector (pCMV-miR302e) increased the teratoma sizes by 140% (250±73 mm³,n=3). Based on that, NTera-2 cells were found to express a moderatelevel of mir-302 (FIG. 8B). Northern blotting also showed that mir-302expression levels in these differently treated teratoma cells negativelycorrelated to the tumor sizes (FIG. 8B), suggesting that modulatingmir-302 expression can effectively control the teratoma growth in vivo.To validate the previous findings in vitro, we performed westernblotting to confirm the co-suppression of G1-checkpoint regulatorsCDK2-cyclins-D1/D2-BMI-1 and the co-activation of core reprogrammingfactors Oct3/4-Sox2-Nanog in the mir-302-treated teratomas (FIG. 8B).The same results were also confirmed by immunohistochemical (IHC)staining of these proteins in teratoma tissue sections (FIG. 8C; Example14). Most noteworthily, we found that mir-302 inhibited teratoma cellgrowth without affecting its nature in pluripotent differentiation,indicating that high concentrated mir-302 plays a dual role as a tumorsuppressor and a reprogramming factor. Based on this dual function ofmir-302 and the consistent data in vitro and in vivo, we conclude thatthe same anti-proliferative mechanism of mir-302 observed in vitro canbe applied to inhibit teratoma growth in vivo, which may serve as apotential treatment for a variety of tumors.

Mir-302 May Cause Cell Senescence Through p16Ink4a/p14ARF ActivationRather than Telomere Shortening

Human iPS cells have been reported to exhibit problems of earlysenescence and limited expansion (Banito et al., 2009; Feng et al.,2010). Normal adult cells also undergo a limited number of divisions andfinally reach a quiescence state called replicative senescence. Cellsthat escape from replicative senescence often become immortal cells suchas tumor/cancer cells; thus, replicative senescence is a normal defensemechanism against tumor/cancer cell formation. In this study, we havefound that mir-302 can directly silence BMI-1 to inducep16Ink4a/p14ARF-associated cell cycle regulation. Other studies havefurther implicated that BMI-1 can also activate human telomerase reversetranscriptase (hTERT) transcription and increase telomerase activity tobypass replicative senescence and increase the cell life span (Dimri etal., 2002). Thus, it is conceivable that mir-302 overexpression maycause hTERT-associated senescence in mirPS cells. To clarify this point,we performed telomeric repeat amplification protocol (TRAP) assay(Example 16) to measure the telomerase activity. Surprisingly, as shownin FIG. 9A, all mirPS cells treated with 10 μM Dox exhibit a strongtelomerase activity similar to that of their original tumor/cancer cellsand hES H1/H9 cells. Moreover, western blotting also showed that hTERTexpression was increased rather than decreased in these mirPS cells(FIG. 9B). The increase of relative telomerase activity was alsoconfirmed by telomerase PCR ELISA assay (FIG. 9C). In addition, wefurther detected the silencing of lysine-specific histone demethylaseAOF2 (also known as KDM1/LSD1) and histone deacetylase HDAC2 in thesemirPS cells (FIG. 9B). Previous studies have reported that AOF2 isrequired for the transcriptional suppression of hTERT and deficiency ofboth AOF2 and HDAC2 induces hTERT overexpression (Won et al., 2002; Zhuet al., 2008). Our recent study has also found that both AOF2 and HDAC2are strong targets of mir-302 and are both silenced in the mirPS cells(FIG. 10). Therefore, mir-302 actually increases telomerase activityrather than causes hTERT-associated senescence in mirPS cells. However,the effect of this increased hTERT activity may be counteracted by themir-302-induced BMI-1 suppression and p16Ink4a/p14ARF activation,resulting in a balance for preventing tumor/cancer cell formation inmirPS cells.

In sum, our present invention utilizes a novel tumor suppressor functionof mir-302 for cancer therapy. We found that mir-302-mediated cell cycleregulation involves a highly coordinate mechanism between co-suppressionof G1-checkpoint regulators and activation of CDK inhibitors. All thesegenetic events must occur simultaneously to prevent any loophole forG1-S progression. Quiescence at G0/G1 phase cell cycle is important forSCR initiation. In this dormant state, somatic cell genomes can belargely demethylated and over 91% of the cellular transcriptome arereprogrammed to a hES-like gene expression pattern. Through decipheringthe interactions between mir-302 and its target genes, we learned theintricate mechanism for mir-302-associated cell cycle regulation duringSCR as shown in FIG. 11. Our previous studies have demonstrated thatmir-302 silences its targeted epigenetic regulators to activateOct3/4-Sox2-Nanog co-expression, and in turn these reprogramming factorsfunction to induce SCR (Lin et al., 2008). In advance, this inventionfurther reveals that mir-302 concurrently silences CDK2, cyclins D1/D2and BMI-1 to attenuate cell division during SCR. Proper control of thecell cycle rate is of critical biological importance in preventing thetumorigenecity of oncogenes that are often activated during SCR. Tothis, mir-302 silences CDK2 and cyclins D1/D2 to hinder the G1-Stransition at this critical moment. Meanwhile, inhibition of BMI-1further enhances the tumor suppressor activities of p16Ink4a and p19Arf.Through these synergistic cell cycle regulation pathways, mir-302 isable to initiate SCR while not aggravating cell tumorigenecity.

Advantageously, there are at least five breakthroughs in the presentinvention. First, one mir-302-like gene effector can replace all fourreprogramming transcription factors Oct4-Sox2-Klf4-c-Myc andOct4-Sox2-Nanog-Lin28 for reprogramming human cells to hES-like stemcells. These reprogrammed cells are useful for stem cell therapy.Second, because of the small size (about 23 ribonucleotides) of amir-302-like gene silencing effector, the vector expressing such a smallsized RNA can be designed to be very compact and highly efficient for invivo transfection. Third, the RNA-related cytotoxicity is prevented byintracellular NMD system and inducible expression. Fourth,mir-302-induced apoptosis only occurs in tumor/cancer cells rather thannormal human cells. Last, the present invention has used polysomal,liposomal and electroporation-based transfection in place ofretroviral/lentiviral infection to deliver the mir-302-expressingnucleic acid composition into tumor/cancer cells, confirming the safetyand therapeutic use of the mir-302-like gene silencing effectors invitro and in vivo. Taken together, these advantages have shown thefeasibility of using the mir-302-like gene silencing effector and itsexpression composition for tumor/cancer therapy, providing a completelynovel design for the development of universal cancer drugs and/orvaccines.

A. DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below:

Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar moiety(pentose), a phosphate, and a nitrogenous heterocyclic base. The base islinked to the sugar moiety via the glycosidic carbon (1′ carbon of thepentose) and that combination of base and sugar is a nucleoside. Anucleoside containing at least one phosphate group bonded to the 3′ or5′ position of the pentose is a nucleotide.

Oligonucleotide: a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

Nucleic Acid: a polymer of nucleotides, either single or doublestranded.

Nucleotide Analog: a purine or pyrimidine nucleotide that differsstructurally from A, T, G, C, or U, but is sufficiently similar tosubstitute for the normal nucleotide in a nucleic acid molecule.

Nucleic Acid Composition: a nucleic acid composition refers topolynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid(RNA) in either single-stranded or double-stranded molecular structures.

Gene: a nucleic acid whose nucleotide sequence codes for an RNA and/or apolypeptide (protein). A gene can be either RNA or DNA.

Base Pair (bp): a partnership of adenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA,uracil (U) is substituted for thymine. Generally the partnership isachieved through hydrogen bonding.

Precursor messenger RNA (pre-mRNA): primary ribonucleotide transcriptsof a gene, which are produced by type-II RNA polymerase (Pol-II)machineries in eukaryotes through an intracellular mechanism termedtranscription. A pre-mRNA sequence contains a 5′-end untranslatedregion, a 3′-end untranslated region, exons and introns.

Intron: a part or parts of a gene transcript sequence encodingnon-protein-reading frames, such as in-frame intron, 5′-untranslatedregion (5′-UTR) and 3′-UTR.

Exon: a part or parts of a gene transcript sequence encodingprotein-reading frames (cDNA), such as cDNA for cellular gene, mammaliangene, embryonic stem cell marker gene, fluorescent protein marker gene,luciferase gene, lac-Z reporter gene, viral gene, jumping gene,transposon, and a combination thereof.

Messenger RNA (mRNA): assembly of pre-mRNA exons, which is formed afterintron removal by intranuclear spliceosomal machineries and served as aprotein-coding RNA for protein synthesis.

cDNA: a single stranded DNA that is complementary to an mRNA sequenceand does not contain any intronic sequences.

Sense: a nucleic acid molecule in the same sequence order andcomposition as the homologous mRNA. The sense conformation is indicatedwith a “+”, “s” or “sense” symbol.

Antisense: a nucleic acid molecule complementary to the respective mRNAmolecule. The antisense conformation is indicated as a “−” symbol orwith an “a” or “antisense” in front of the DNA or RNA, e.g., “aDNA” or“aRNA”.

5′-end: a terminus lacking a nucleotide at the 5′ position of successivenucleotides in which the 5′-hydroxyl group of one nucleotide is joinedto the 3′-hydroyl group of the next nucleotide by a phosphodiesterlinkage. Other groups, such as one or more phosphates, may be present onthe terminus.

3′-end: a terminus lacking a nucleotide at the 3′ position of successivenucleotides in which the 5′-hydroxyl group of one nucleotide is joinedto the 3′-hydroyl group of the next nucleotide by a phosphodiesterlinkage. Other groups, most often a hydroxyl group, may be present onthe terminus.

Template: a nucleic acid molecule being copied by a nucleic acidpolymerase. A template can be single-stranded, double-stranded orpartially double-stranded, depending on the polymerase. The synthesizedcopy is complementary to the template, or to at least one strand of adouble-stranded or partially double-stranded template. Both RNA and DNAare synthesized in the 5′ to 3′ direction. The two strands of a nucleicacid duplex are always aligned so that the 5′ ends of the two strandsare at opposite ends of the duplex (and, by necessity, so then are the3′ ends).

Nucleic Acid Template: a double-stranded DNA molecule, double strandedRNA molecule, hybrid molecules such as DNA-RNA or RNA-DNA hybrid, orsingle-stranded DNA or RNA molecule.

Conserved: a nucleotide sequence is conserved with respect to apre-selected (referenced) sequence if it non-randomly hybridizes to anexact complement of the pre-selected sequence.

Complementary or Complementarity or Complementation: used in referenceto polynucleotides (i.e. a sequence of nucleotides) related by thebase-pairing rules. For example, the sequence “A-G-T” is complementaryto the sequence “T-C-A,” and also to “T-C-U.” Complementation can bebetween two DNA strands, a DNA and an RNA strand, or between two RNAstrands. Complementarity may be “partial” or “complete” or “total”.Partial complementarity or complementation occurs when only some of thenucleic acid bases are matched according to the base pairing rules.Complete or total complementarity or complementation occurs when thebases are completely matched between the nucleic acid strands. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, as well as in detection methods that depend on bindingbetween nucleic acids. Percent complementarity or complementation refersto the number of mismatch bases over the total bases in one strand ofthe nucleic acid. Thus, a 50% complementation means that half of thebases were mismatched and half were matched. Two strands of nucleic acidcan be complementary even though the two strands differ in the number ofbases. In this situation, the complementation occurs between the portionof the longer strand corresponding to the bases on that strand that pairwith the bases on the shorter strand.

Homologous or Homology: refers to a polynucleotide sequence havingsimilarities with a gene or mRNA sequence. A nucleic acid sequence maybe partially or completely homologous to a particular gene or mRNAsequence, for example. Homology may also be expressed as a percentagedetermined by the number of similar nucleotides over the total number ofnucleotides.

Complementary Bases: nucleotides that normally pair up when DNA or RNAadopts a double stranded configuration.

Complementary Nucleotide Sequence: a sequence of nucleotides in asingle-stranded molecule of DNA or RNA that is sufficientlycomplementary to that on another single strand to specifically hybridizebetween the two strands with consequent hydrogen bonding.

Hybridize and Hybridization: the formation of duplexes betweennucleotide sequences which are sufficiently complementary to formcomplexes via base pairing. Where a primer (or splice template)“hybridizes” with target (template), such complexes (or hybrids) aresufficiently stable to serve the priming function required by a DNApolymerase to initiate DNA synthesis. There is a specific, i.e.non-random, interaction between two complementary polynucleotides thatcan be competitively inhibited.

Posttranscriptional Gene Silencing: a targeted gene knockout orknockdown effect at the level of mRNA degradation or translationalsuppression, which is usually triggered by either foreign/viral DNAtransgenes or small inhibitory RNAs.

RNA Interference (RNAi): a posttranscriptional gene silencing mechanismin eukaryotes, which can be triggered by small inhibitory RNA moleculessuch as microRNA (miRNA), small hairpin RNA (shRNA) and smallinterfering RNA (siRNA). These small RNA molecules usually function asgene silencers, interfering with expression of intracellular genescontaining either completely or partially complementarity to the smallRNAs.

Non-coding RNA: an RNA transcript that cannot be used to synthesizepeptides or proteins through intracellular translation machineries.

MicroRNA (miRNA): single-stranded RNAs capable of binding to targetedgene transcripts that have partial complementarity to the miRNA. MiRNAis usually about 17-27 oligonucleotides in length and is able to eitherdirectly degrade its intracellular mRNA target(s) or suppress theprotein translation of its targeted mRNA, depending on thecomplementarity between the miRNA and its target mRNA. Natural miRNAsare found in almost all eukaryotes, functioning as a defense againstviral infections and allowing regulation of gene expression duringdevelopment of plants and animals.

Pre-miRNA: hairpin-like single-stranded RNAs containing stem-arm andstem-loop regions for interacting with intracellular RNaseIIIendoribonucleases to produce one or multiple microRNAs (miRNAs) capableof silencing a targeted gene or genes complementary to the microRNAsequence(s). The stem-arm of a pre-miRNA can form either a perfectly(100%) or a partially (mis-matched) hybrid duplexes, while the stem-loopconnects one end of the stem-arm duplex to form a circle or hairpin-loopconformation.

Small interfering RNA (siRNA): short double-stranded RNAs sized about18-25 perfectly base-paired ribonucleotide duplexes and capable ofdegrading target gene transcripts with almost perfect complementarity.

Small or short hairpin RNA (shRNA): single-stranded RNAs that contain apair of partially or completely matched stem-arm nucleotide sequencesdivided by an unmatched loop oligonucleotide to form a hairpin-likestructure. Many natural miRNAs are derived from hairpin-like RNAprecursors, namely precursor microRNA (pre-miRNA).

Vector: a recombinant nucleic acid composition such as recombinant DNA(rDNA) capable of movement and residence in different geneticenvironments. Generally, another nucleic acid is operatively linkedtherein. The vector can be capable of autonomous replication in a cellin which case the vector and the attached segment is replicated. Onetype of preferred vector is an episome, i.e., a nucleic acid moleculecapable of extrachromosomal replication. Preferred vectors are thosecapable of autonomous replication and expression of nucleic acids.Vectors capable of directing the expression of genes encoding for one ormore polypeptides and/or non-coding RNAs are referred to herein as“expression vectors”. Particularly important vectors allow cloning ofcDNA from mRNAs produced using a reverse transcriptase. A vector maycontain components consisting of a viral or a type-II RNA polymerase(Pol-II) promoter, or both, a Kozak consensus translation initiationsite, polyadenylation signals, a plurality of restriction/cloning sites,a pUC origin of replication, a SV40 early promoter for expressing atleast an antibiotic resistance gene in replication-competent prokaryoticcells, an optional SV40 origin for replication in mammalian cells,and/or a tetracycline responsive element.

Cistron: a sequence of nucleotides in a DNA molecule coding for an aminoacid residue sequence and including upstream and downstream DNAexpression control elements.

Promoter: a nucleic acid to which a polymerase molecule recognizes,perhaps binds to, and initiates synthesis. For the purposes of theinstant invention, a promoter can be a known polymerase binding site, anenhancer and the like, any sequence that can initiate synthesis by adesired polymerase.

Antibody: a peptide or protein molecule having a pre-selected conserveddomain structure coding for a receptor capable of binding a pre-selectedligand.

Primary RNA Transcript: an ribonucleotide sequence selected from thegroup consisting of mRNA, hnRNA, rRNA, tRNA, snoRNA, snRNA,pre-microRNA, viral RNA and their RNA precursors as well as derivatives.

Intron Excision: a cellular mechanism responsible for RNA processing,maturation and degradation, including RNA splicing, exosome digestion,nonsense-mediated decay (NMD) processing, and a combination thereof.

Donor Splice Site: a nucleic acid sequence either containing orhomologous to the SEQ.ID.NO.4 sequence or 5′-GTAAG-3′.

Acceptor Splice Site: a nucleic acid sequence either containing orhomologous to the SEQ.ID.NO.5 sequence or 5′-CTGCAG-3′.

Branch Point: an adenosine (A) nucleotide located within a nucleic acidsequence containing or homologous to the SEQ.ID.NO.6 sequence or5′-TACTAAC-3′.

Poly-Pyrimidine Tract: a high T or C content nucleic acid sequencecontaining or homologous to the SEQ.ID.NO.7 or SEQ.ID.NO.8 sequence.

Targeted Cell: a single or a plurality of human cells selected from thegroup consisting of a somatic cell, a tissue, a stem cell, a germ-linecell, a teratoma cell, a tumor cell, a cancer cell, and a combinationthereof.

Cancerous Tissue: a neoplastic tissue derived from the group consistingof skin cancer, prostate cancer, breast cancer, liver cancer, lungcancer, brain tumor/cancer, lymphoma, leukemia and a combinationthereof.

Expression-Competent Vector: a linear or circular form of single- ordouble-stranded DNA selected form the group consisting of plasmid, viralvector, transposon, retrotransposon, DNA transgene, jumping gene, and acombination thereof.

Antibiotic Resistance Gene: a gene capable of degrading antibioticsselected from the group consisted of penicillin G, ampicillin, neomycin,G418, paromycin, kanamycin, streptomycin, erythromycin, spectromycin,phophomycin, tetracycline, rifapicin, amphotericin B, gentamycin,chloramphenicol, cephalothin, tylosin, and a combination thereof.

Type-II RNA Polymerase Equivalent: a transcription machinery selectedfrom the group consisting of type-II (Pol-II), type-III (Pol-III),type-I (Pol-I), and viral RNA polymerases.

Restriction/Cloning Site: a DNA motif for restriction enzyme cleavageincluding but not limited AatII, AccI, AflII/III, AgeI, ApaI/LI, AseI,Asp718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I, BspHI/LU11I/120I,BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, ClaI, Csp6I, DpnI, DraI/II, EagI,Ecl136II, EcoRI/RII/47III/RV, EheI, FspI, HaeIII, HhaI, HinPI, HindIII,HinfI, HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI, MseI, NaeI,NarI, NcoI, NdeI, NgoMI, NotI, NruI, NsiI, PmII, Ppu10I, PstI, PvuI/II,RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI,XbaI, XhoI, XmaI cleavage site.

Gene Delivery: a genetic engineering method selected from the groupconsisting of polysomal transfection, liposomal transfection, chemicaltransfection, electroporation, viral infection, DNA recombination,transposon insertion, jumping gene insertion, microinjection, gene-gunpenetration, and a combination thereof.

Genetic Engineering: a DNA recombination method selected from the groupconsisting of DNA restriction and ligation, homologous recombination,transgene incorporation, transposon insertion, jumping gene integration,retroviral infection, and a combination thereof.

Cell Cycle Regulator: a cellular gene involved in controlling celldivision and proliferation rates, consisting but not limited of CDK2,CDK4, CDK6, cyclins, BMI-1, p14/p19Arf, p15Ink4b, p16Ink4a, p18Ink4c,p21Cip1/Waf1, and p27Kip1, and a combination thereof.

Tumor Suppression: a cellular anti-tumor and anti-cancer mechanismconsisting but not limited of cell cycle attenuation, G0/G1-checkpointarrest, tumor suppression, anti-tumorigenecity, cancer cell apoptosis,and a combination thereof.

Gene Silencing Effect: a cell response after a gene function issuppressed, consisting but not limited of cell cycle attenuation,G0/G1-checkpoint arrest, tumor suppression, anti-tumorigenecity, cancercell apoptosis, and a combination thereof.

B. COMPOSITIONS AND METHODS

A design and method for using a recombinant nucleic acid compositioncapable of being delivered, transcribed and processed into mir-302-likegene silencing effectors in targeted human cells and thus inducingspecific gene silencing effects on mir-302-targeted cell cycleregulators and oncogenes in the cells, comprising the steps of:

-   a) providing a recombinant nucleic acid composition capable of being    delivered, transcribed and processed into at least a gene silencing    effector interfering a plurality of cellular genes targeted by    mir-302; and-   b) treating a cell substrate with said recombinant nucleic acid    composition.

The above recombinant nucleic acid composition, further comprises:

-   a) A plurality of exons, wherein said exons can be linked to form a    gene transcript possessing a desired function; and-   b) At least an intron, wherein said intron contains a recombinant    mir-302 homologue and can be cleaved out of the exons through    intracellular RNA splicing and processing mechanisms.

The intron of the above recombinant nucleic acid composition, furthercomprises:

-   a) A 5′-donor splice site for spliceosomal binding;-   b) A gene-silencing effector insert homologous to members of the    mir-302 family;-   c) A branch point motif for spliceosomal recognition;-   d) A poly-pyrimidine tract for spliceosomal interaction;-   e) A 3′-acceptor splice site for spliceosomal binding; and-   f) A plurality of linkers for connecting each of the above    components in a 5′ to 3′ direction.

Preferably, the present invention has adopted a novel design andstrategy for either inducible or constitutive expression of mir-302-likegene silencing effectors in the transfected cells. Mir-302-like genesilencing effectors include mir-302a, mir-302b, mir-302c, mir-302d, andtheir hairpin-like microRNA precursors (pre-miRNAs) as well as manuallyre-designed small hairpin RNA (shRNA) homologues/derivatives, and acombination thereof. The transcription of mir-302-like gene silencingeffectors is driven either by a constitutive (i.e. CMV) ordrug-inducible (i.e. TRE-CMV) promoter. Preferably, the drug-induciblerecombinant nucleic acid composition is a Tet-On vector containing arecombinant transgene inserted with either a recombinant mir-302 familycluster (mir-302s; hybrid of SEQ.ID.NOs.9-16) or a manually re-designedmir-302 shRNA homologue (i.e. hybrid of SEQ.ID.NOs.17 and 18). The cellsubstrate may express the mir-302 target genes either in vitro, ex vivoor in vivo. By silencing the mir-302-targeted cell cycle regulators andoncogenes, the present invention is able to suppress cell tumorigenecityand reprogram the treated cells into non-tumor/cancer cells.

EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (molar); mM (millimolar); μm (micromolar); mol(moles); pmol (picomolar); gm (grams); mg (milligrams); μg (micrograms);ng (nanograms); L (liters); ml (milliliters); μl (microliters); ° C.(degrees Centigrade); cDNA (copy or complementary DNA); DNA(deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA(double-stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA(ribonucleic acid); PBS (phosphate buffered saline); NaCl (sodiumchloride); HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid);HBS (HEPES buffered saline); SDS (sodium dodecylsulfate); Tris-HCl(tris-hydroxymethylaminomethane-hydrochloride); ATCC (American TypeCulture Collection, Rockville, Md.); hES (human embryonic stem cells);iPS (induced pluripotent stem cells); and SCR (somatic cellreprogramming).

Example 1 Cell Culture and Transfection

Human cancer NTera-2, HepG2, MCF7, PC3 and Colo829 cell lines wereacquired from ATCC, while human hair follicle cells (hHFCs) wereisolated and dissociated from a minimum of two hair dermal papillae by 4mg/ml collagenase I digestion for 45 min in fresh RPMI 1640 mediumsupplemented with 20% FBS. For culturing melanocytes, the isolated cellswere cultivated in Medium 254 with the addition of human melanocytesgrowth supplement-2 (HMGS-2, Invitrogen, Carlsbad, Calif.) in theabsence of antibiotics at 37° C. under 5% CO₂. Cultures were passaged at70%-80% confluency by exposing cells to trypsin/EDTA solution for 1 minand rinsing once with phenol red-free DMEM medium (Invitrogen), and thedetached cells were replated at 1:10 dilution in fresh Medium 254 withHMGS-2 supplements. For electroporation, a mixture ofpTet-On-tTS-mir302s (10 μg) and pTet-On-Adv-Neo(−) (50 μg) was addedwith the isolated cells (20,000-50,000) in a hypoosmolar buffer (200 μl;Eppendorf, Westbury, N.Y.) and electroporation was performed usingEppendorf Multiporator at 300-400 volts for 150 μsec. The electroporatedcells were first grown in phenol red-free DMEM medium (Invitrogen)supplemented with 20% knockout serum, 1% MEM nonessential amino acids,10 ng/ml bFGF, 1 mM GlutaMax, and 1 mM sodium pyruvate, for 24 hours at37° C. under 5% CO₂. Then, 850 μg/ml G418 and >3.75 μg/ml doxycycline(Dox) were added and refreshed daily for 3-5 days till the cellsexpressed strong red fluorescent RGFP. Next, the individual redfluorescent cell (mirPS) was monitored under a TE2000 invertedmicroscopic system (Nikon, Melville, N.Y.) and separately collected intoa 96-well, using MO-188NE 3D micromanipulators (Nikon). In the absenceof Dox, the mirPS cells were grown and passaged in knockout DMEM/F-12medium (Invitrogen) supplemented with 20% knockout serum, 1% MEMnonessential amino acids, 100 μM β-mercaptoethanol, 1 mM GlutaMax, 1 mMsodium pyruvate, 10 ng/ml bFGF, 100 IU/ml penicillin/100 μg/mlstreptomycin/250 μg/ml G418, 0.1 μM A83-01, and 0.1 μM valproic acid(Stemgent, San Diego, Calif.), at 37° C. under 5% CO₂. Alternatively, inthe presence of Dox (3.75-5 μg/ml; Sigma-Aldrich, St. Louis, Mo.), themirPS cells were cultivated and passaged in the same feeder-freecultural condition with addition of 0.05 μM GSK inhibitor SB216763(Stemgent). Addition of GSK inhibitor could facilitate mirPS cellproliferation but with a slight tendency to cause neuraldifferentiation. For neural cell induction, the mirPS cells were grownin the above feeder-free cultural condition with 0.05 μM SB216763 but noDox.

Example 2 Construction of Recombinant Vectors Expressing mir-302s

The mir-302 familial cluster (mir-302s) was generated as reported (Linet al., 2008). The mir-302s cluster consists of four parts, includingprecursor miRNAs (pre-miRNAs) of mir-302a, b, c, and d. Syntheticoligonucleotides (Sigma-Genosys, St. Louis, Mo.) used for constructingthe mir-302s cluster were listed below. For expression, we mixed anequal amount (1:1) of the mir-302s cluster and a pre-made SpRNAi-RGFPrecombinant gene (Lin et al., 2006 and 2008), and then digested themixture with MluI/PvuI restriction enzymes at 37° C. for 4 hours. Thedigested mixture was collected with a gel extraction filter (Qiagen, CA)in 30 μl of ddH₂O and ligated together using T4 DNA ligase at 8° C. for16 hours. This formed a recombinant mir-302-expressing SpRNAi-RGFP gene,which was further cleaved with XhoI/HindIII restriction enzymes andinserted into a Dox-inducible pSingle-tTS-shRNA vector (Clontech, PaloAlto, Calif.). This formed an inducible pTet-On-tTS-mir302s expressionvector. Then, we further modified the pTet-On-tTS-mir302s vector byreplacing its U6 promoter with a TRE-CMV promoter isolated from apTRE-Tight plasmid (Clontech). For generating a non-inducible,constitutive pCMV-miR302s expression vector, we cleaved the modifiedpTet-On-tTS-mir302s vector with EcoR1 restriction enzyme, removed theupstream tTS-TRE sequence (1.5 kb) by gel electrophoresis, and recoveredthe cleaved vector from the gel for further DNA ligation to complete theformation of the non-inducible pCMV-miR302s vector.

Synthetic oligonucleotides for DNA recombination of the mir-302 familialpre-miRNA cluster were listed as follows: mir-302a-sense, 5′-GTCACGCGTTCCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTTTTGGTGATGG ATAGATCTCT C-3′ (SEQ.ID.NO.9); mir-302a-antisense,5′-GAGAGATCTA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTACATCCACGTT TAAGTGGTGG GAACGCGTGA C-3′ (SEQ.ID.NO.10); mir-302b-sense,5′-ATAGATCTCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAAGTGCTTCCAT GTTTTAGTAG GAGTCGCTCA TATGA-3′ (SEQ.ID.NO.11);mir-302b-antisense, 5′-TCATATGAGC GACTCCTACT AAAACATGGA AGCACTTACTTTCAAAGTCA CAGAAAGCAC TTCCATGTTA AAGTTGAAGG GAGCGAGAGA TCTAT-3′(SEQ.ID.NO.12); mir-302c-sense, 5′-CCATATGGCT ACCTTTGCTT TAACATGGAGGTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3′(SEQ.ID.NO.13); mir-302c-antisense, 5′-ATGTCTAGAC GCCTCCACTG AAACATGGAAGCACTTACTT CTGTTTCACA CAGCAGGTAC CTCCATGTTA AAGCAAAGGT AGCCATATGG-3′(SEQ.ID.NO.14); mir-302d-sense, 5′-CGTCTAGACA TAACACTCAA ACATGGAAGCACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGCGATCGC AT-3′(SEQ.ID.NO.15); and mir-302d-antisense, 5′-ATGCGATCGC GAACACTCAAACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3′(SEQ.ID.NO.16). Alternatively, we used a manually re-designed shRNAformed by the hybrid of synthetic miR-302s-sense, 5′-GCAGATCTCGAGGTACCGAC GCGTCCTCTT TACTTTAACA TGGAAATTAA GTGCTTCCAT GTTTGAGTGGTGTGGCGCGA TCGATATCTC TAGAGGATCC ACATC-3′ (SEQ.ID.NO.17) andmir-302s-antisense, 5′-GATGTGGATC CTCTAGAGAT ATCGATCGCG CCACACCACTCAAACATGGA AGCACTTAAT TTCCATGTTA AAGTAAAGAG GACGCGTCGG TACCTCGAGATCTGC-3′ (SEQ.ID.NO.18), in place of the mir-302 pre-miRNA cluster foreasy intronic insertion. In design of mir-302 homologues, thymine (T)can be used in place of uracil (U) or vice versa. All these syntheticsequences were purified with PAGE gel extraction before ligation.

The recombinant mir-302 familial pre-miRNA cluster (mir-302s) was formedby linkage of four mir-302a-d hybrids, including mir-302a-sense andmir-302a-antisense, mir-302b-sense and mir-302b-antisense,mir-302c-sense and mir-302c-antisense, and mir-302d-sense andmir-302d-antisense. The hybrids of mir-302a, mir-302b, mir-302c, andmir-302d were digested by PvuI/XhoI, XhoI/NheI, NheI/XbaI, and XbaI/MluIrestriction enzymes, respectively, and collected together by a gelextraction filter column in 35 μl autoclaved ddH₂O (Qiagen, CA).Immediately after that, the mixed hybrids were ligated to form a clusterwith T4 DNA ligase (Roche, 20U) and further inserted into thePvuI/MluI-linearized SpRNAi-RGFP recombinant gene. Alternatively, themir-302 shRNA made by hybridizing SEQ.ID.NO.17 and SEQ.ID.NO.18 wascleaved with PvuI/MluI restriction enzymes and inserted into thePvuI/MluI-linearized SpRNAi-RGFP.

The pTet-On-tTS-mir302s and CMV-mir302s vectors were propagated in E.coli DH5α LB culture containing 100 μg/ml ampicillin (Sigma Chemical,St. Louis, Mo.). The propagated pTet-On-tTS-mir302s and CMV-mir302svectors were isolated and purified using an Endo-Free Maxi-Prep PlasmidExtraction Kit (Qiagen, CA).

Example 3 MicroRNA (miRNA) Microarray Analysis

At 70% confluency, small RNAs from each cell culture were isolated,using the mirVana™ miRNA isolation kit (Ambion). The purity and quantityof the isolated small RNAs were assessed, using 1% formaldehyde-agarosegel electrophoresis and spectrophotometer measurement (Bio-Rad), andthen immediately frozen in dry ice and submitted to LC Sciences (SanDiego, Calif.) for miRNA microarray analysis. Each microarray chip washybridized a single sample labeled with either Cy3 or Cy5 or a pair ofsamples labeled with Cy3 and Cy5, respectively. Background subtractionand normalization were performed. For a dual sample assay, a p-valuecalculation was performed and a list of differentially expressedtranscripts more than 3-fold was produced. The result was shown in FIG.1C.

Example 4 Northern Blot Analysis

Total RNAs (10 μg) were isolated with a mirVana™ miRNA Isolation Kit(Ambion, Austin, Tex.), fractionated by either 15% TBE-ureapolyacrylamide gel or 3.5% low melting point agarose gelelectrophoresis, and electroblotted onto a nylon membrane. Detection ofmir-302 was performed with a [LNA]-DNA probe (5′-[TCACTGAAAC] ATGGAAGCACTTA-3′) (SEQ.ID.NO.19), while probes for other gene detection weresynthesized and listed in Table 1. All probes were purified byhigh-performance liquid chromatography (HPLC) and tail-labeled withterminal transferase (20 units) for 20 min in the presence of [³²P]-dATP(>3000 Ci/mM, Amersham International, Arlington Heights, Ill.).Hybridization was carried out in the mixture of 50% freshly deionizedformamide (pH 7.0), 5×Denhardt's solution, 0.5% SDS, 4×SSPE and 250mg/mL denatured salmon sperm DNA fragments (18 hr, 42° C.). Membraneswere sequentially washed twice in 2×SSC, 0.1% SDS (15 min, 25° C.), andonce in 0.2×SSC, 0.1% SDS (45 min, 37° C.) before autoradiography. Theresults were shown in FIGS. 1D, 4A and 8B.

Example 5 Western Blot Analysis

Cells (10⁶) were lysed with a CelLytic-M lysis/extraction reagent(Sigma) supplemented with protease inhibitors, Leupeptin, TLCK, TAME andPMSF, following the manufacturer's suggestion. Lysates were centrifugedat 12,000 rpm for 20 min at 4° C. and the supernatant was recovered.Protein concentrations were measured using an improved SOFTmax proteinassay package on an E-max microplate reader (Molecular Devices, Calif.).Each 30 μg of cell lysate was added to SDS-PAGE sample buffer underreducing (+50 mM DTT) and non-reducing (no DTT) conditions, and boiledfor 3 min before loading onto a 6˜8% polyacylamide gel. Proteins wereresolved by SDS-polyacrylamide gel electrophoresis (PAGE),electroblotted onto a nitrocellulose membrane and incubated in Odysseyblocking reagent (Li-Cor Biosciences, Lincoln, NB) for 2 hours at roomtemperature. Then, a primary antibody was applied to the reagent andincubated the mixture at 4° C. Primary antibodies included Oct3/4 (SantaCruz Biotechnology, Santa Cruz, Calif.), Sox2 (Santa Cruz), Nanog (SantaCruz), Lin28 (Abcam Inc., Cambridge, Mass.), UTF1 (Abcam), Klf4 (SantaCruz), TRP1 (Santa Cruz), keratin 16 (Abcam), CDK2 (Santa Cruz), cyclinD1 (Santa Cruz), cyclin D2 (Abcam), BMI-1 (Santa Cruz), AOF2 (Sigma),HDAC2 (Abcam), hTERT (Santa Cruz), 8-actin (Chemicon, Temecula, Calif.),and RGFP (Clontech). After overnight, the membrane was rinsed threetimes with TBS-T and then exposed to goat anti-mouse IgG conjugatedsecondary antibody to Alexa Fluor 680 reactive dye (1:2,000;Invitrogen-Molecular Probes), for 1 hour at the room temperature. Afterthree additional TBS-T rinses, fluorescent scanning of the immunoblotand image analysis were conducted using Li-Cor Odyssey Infrared Imagerand Odyssey Software v.10 (Li-Cor). The results were shown in FIGS. 1D,4B, 7C, 7D, 8B and 9B.

Example 6 Apoptotic DNA Laddering Assay

Genomic DNAs were isolated from about two million cells using anApoptotic DNA Ladder Kit (Roche Biochemicals, Indianapolis, Iowa) and 2μg of the isolated DNAs were further assessed by 2% agarose gelelectrophoresis, according to the manufacturers' suggestion. The resultwas shown in FIG. 1F.

Example 7 DNA-Density Flow Cytometry

Cells were trypsinized, pelleted and fixed by re-suspension in 1 ml ofpre-chilled 70% methanol in PBS for 1 hour at −20° C. The cells werepelleted and washed once with 1 ml of PBS. The cells were pelleted againand resuspended in 1 ml of 1 mg/ml propidium iodide, 0.5 μg/ml RNase inPBS for 30 min at 37° C. Approximately 15,000 cells were then analyzedon a BD FACSCalibur (San Jose, Calif.). Cell doublets were excluded byplotting pulse width versus pulse area and gating on the single cells.The collected data were analyzed using the software package Flowjo usingthe “Watson Pragmatic” algorithm. The result was shown in FIGS. 3A-B,6A-B and 6C.

Example 8 Genome-Wide Microarray Analysis

Human genome GeneChip U133 plus 2.0 arrays (Affymetrix, Santa Clara,Calif.) were used to detect the alterations of over 47,000 human geneexpression patterns in tested cells. Total RNAs from each tested samplewere isolated using a mirVana™ miRNA Isolation Kit (Ambion), followingthe manufacturer's suggestion. The purity and quantity of isolated RNAswere assessed using 1% formaldehyde-agarose gel electrophoresis andspectrophotometer measurement (Bio-Rad). The sample signals werenormalized using the total average difference between perfectly matchedprobes and mismatched probes. Alterations of genome-wide gene expressionpatterns were analyzed using Affymetrix Microarray Suite version 5.0,Expression Console™ version 1.1.1 (Affymetrix) and Genesprings (SiliconGenetics) softwares. Changes in gene expression rates more than 1-foldwere considered as positive differential genes. For gene clustering, aplug-in program Genetrix (Epicenter Software) was used in conjunctionwith the Affymetrix softwares. Signals of the sample were normalizedwith the internal house-keeping control average in each microarray. Theresult of scatter plot analysis was shown in FIG. 5A.

Example 9 DNA Demethylation Assays

Genomic DNAs were isolated from about two million cells using a DNAIsolation Kit (Roche) and 1 μg of the isolated DNAs were further treatedwith bisulfite (CpGenome DNA modification kit, Chemicon, Temecula,Calif.), according to the manufacturers' suggestions. Meanwhile, 2 μg ofuntreated DNAs were digested with a CCGG-cutting restriction enzymeHpaII and then analyzed by 1% agarose gel electrophoresis to determinegenome-wide demethylation (FIG. 5B). The treatment with bisulfiteconverted all unmethylated cytosine to uracil, while methylated cytosineremained as cytosine. For bisulfite DNA sequencing analyses, weamplified the promoter regions of Oct3/4 and Nanog with PCR. Primersincluded 5′-GAGGCTGGAG CAGAAGGATT GCTTTGG-3′ (SEQ.ID.NO.20) and5′-CCCTCCTGAC CCATCACCTC CACCACC-3′ (SEQ.ID.NO.21) for Oct3/4, and5′-TGGTTAGGTT GGTTTTAAAT TTTTG-3′ (SEQ.ID.NO.22) and 5′-AACCCACCCTTATAAATTCT CAATTA-3′ (SEQ.ID.NO.23) for Nanog. The bisulfite-modifiedDNAs (50 ng) were first mixed with the primers (total 100 pmole) in1×PCR buffer, heated to 94° C. for 2 min, and immediately cooled on ice.Next, 25 cycles of PCR were performed as follows: 94° C. for 1 min and70° C. for 3 min, using an Expand High Fidelity PCR kit (Roche). Theamplified DNA product with a correct size was further fractionized by 3%agarose gel electrophoresis, purified with a gel extraction filter(Qiagen), and then used in DNA sequencing. A detailed profile of the DNAmethylation sites was generated by comparing the unchanged cytosine inthe converted DNA sequence to the unconverted one. The result was shownin FIG. 5C.

Example 10 Implantation and Teratoma Formation

Approximately 5-10 mirPS cell-derived embryoid bodies (4- to8-cell-stage) were suspended in 50 μl of a mixture of DMEM and Matrigel(2:1), followed by implantation into the uterus of a 6-week-old femalepseudopregnant immunocompromised SCID-beige mouse. The pseudopregnantmice were prepared by intraperitoneal injection of 1IU human menopausalgonadotrophin (HMG) for two days and then human chorionic gonadotrophin(hCG) for one more day. The cells and mice were not treated with Doxbefore or after implantation. The mice were anesthetized with 2.5%Avertin solution, 0.4 ml per mouse during implantation. Xenograftedmasses were monitored 3-4 weeks after the implantation or when the sizeswere grown to over 100 mm³. Cysts/teratomas were dissected and thevolumes were calculated using the formula (length×width²)/2.Cyst/teratoma lesions were counted, weighed and subjected to furtherhistological analysis. Formation of teratoma-like tissue cysts wasusually observed at approximately 2.5-week post-implantation. The resultwas shown in FIG. 5D.

Example 11 Cell Invasion Assay

Chamber inserts (12-μm pore size, Chemicon) were coated with 200 μg/mlof Matrigel alone or supplemented with 20% FBS in phenol red-free-DMEMwith 1% L-glutamine and dried overnight under sterile conditions. Cellswere harvested, washed, and resuspended in phenol red-free-DMEM to givea final cell density of 1×10⁵ cells/ml. Five hundred microliters of theresulting cell suspension was then dispensed into the top chamberwhereas DMEM conditioned medium (1.5 ml) was added to the bottom chamberto create a chemotactic gradient. Invasion was measured after overnightincubation at 37° C. for 16 hour. Top chambers were wiped with cottonwool, and invading cells on the underside of the membrane were fixed in100% methanol for 10 min, air dried, stained in cresyl violet for 20min, and gently rinsed in water. When dry, the cresyl violet stain onmembranes was eluted using a 100% ethanol/0.2 M NaCitrate (1:1) wash for20 min and absorbance read at 570 nm using a Precision Microplate Reader(Molecular Dynamics). The percentage of invading cells was calculated bycomparison of absorbance in test samples against absorbance determinedon membrane inserts that were not wiped (total cells). The result wasshown in FIG. 6D.

Example 12 Cell Adhesion Assay

Cell Adhesion assay was performed as reported (Lin et al., 2007). Humanbone marrow endothelial cells (hBMECs) were seeded at a density of 1×10⁵cells/ml in 96-well plates and washed with adhesion medium [RPMI1640/0.1% BSA/20 mM HEPES (pH7.4)] before assays. Tested cells weretrypsinized (tumor/cancer cells) or collagenase-digested (mirPS cells),washed in sterile saline, and resuspended at 1×10⁶ cells/ml in PBS with10 μM fura-4 acetoxymethyl ester (fluorescent probe, Sigma) for 1 hourat 37° C. in the dark. The cells were then pelleted, washed inserum-free medium containing 1% (v/v) of probenecid (100 mM) andincubated for 20 min in adhesion medium at 37° C. in the dark toactivate the intracellular fluorescent probe. After that, 10⁵ cells (in300-0 cell suspension/well) were added to the confluent hBMECendothelial monolayer and incubated for 50 min at 37° C. Non-adherentcells were removed using 2×250 μl washes of adhesion medium. Plates wereread in a fluorescent plate reader (Molecular Dynamics) at 37° C. usingan excitation wavelength of 485 nm and an emission wavelength of 530 nm.The result was shown in FIG. 6E.

Example 13 In Vivo Tumorigenecity Assay

The inventors xenografted NTera-2 cells (2×10⁶ cells in a total volumeof 100 μl Matrigel-PBS) into the flanks (e.g. right hind limb) ofeight-week-old male mice (BALB/c nu/nu strain). Tumors were monitoredweekly and in situ injection of pCMV-miR302s vector or pCMV-miR302d* wasconducted one week after the NTera-2 xenograft. Five treatments(three-day intervals for each treatment) of 2 μg PEI-formulatedpCMV-miR302s or pCMV-miR302d* vector (total 10 μg) per g mouse weightwere performed. In vivo-jetPEI Delivery Reagent (Polyplus-transfectionInc., New York, N.Y.) was used as the manufacturer's suggestion. Sampleswere collected either three weeks post injection or when untreatedtumors grew to an average size of approximately 100 mm³. Major organs,such as the blood, brain, heart lung, liver, kidney and spleen, and thexenografts were removed for histological evaluation of tumor lesions andimmunoreactive cytotoxicity. Tumor formation was monitored by palpationand tumor volume was calculated using the formula (length×width²)/2.Tumor lesions were counted, dissected, weighed, and subjected tohistological examination using H&E and immunostaining assays.Histological examination showed no detectable tissue lesions in brain,heart, lung, liver, kidney and spleen. The result was shown in FIG. 8A.

Example 14 Immunostaining Assay

Tissue samples were fixed in 4% paraformaldehyde overnight at 4° C. Thesamples were washed sequentially with 1×PBS, methanol, isopropanol andtetrahydronaphthalene before embedded in paraffin wax. The embeddedsamples were then cut on a microtome at 7-10 μm thickness and mounted onclean TESPA-coated slides. Then, the slides were dewaxed with xylene andmounted under coverslips using mounting media (Richard Allan Scientific,Kalamazoo, Mich.) and stained by hematoxylin and eosin (H&E, Sigma) formorphological observation. Immunohistochemical (IHC) Staining Kits werepurchased from Imgenex (San Diego, Calif.). Processes for antibodydilution and immunostaining were performed according to themanufacturers' suggestions. Primary antibodies included Oct3/4 (SantaCruz), Sox2 (Santa Cruz), Nanog (Santa Cruz), CDK2 (Santa Cruz), cyclinD1 (Santa Cruz), cyclin D2 (Abcam), BMI-1 (Santa Cruz), and RGFP(Clontech). Secondary antibodies used were biotinylated goat anti-rabbitor biotinylated horse anti-mouse antibodies (Chemicon, Temecula,Calif.). Streptavidin-HRP was added as the tertiary antibody. After theslides were washed twice with PBT, the bound antibody was detected usingDAB substrates. Positive results were observed under a 100× microscopewith whole field scanning and measured at 200× magnification forquantitative analysis using a Metamorph Imaging program (Nikon 80imicroscopic quantitation system). The result of scatter plot analysiswas shown in FIG. 8C.

Example 15 Luciferase 3′-UTR Reporter Assay

Luciferase assays were performed using a modified pMir-Report miRNAExpression Reporter Vector System (Ambion). The mir-302 target sites(normal and/or mutant) were inserted in the 3′-UTR cloning site of thepMir-Report Luciferase Reporter vector. The two target sites weresynthesized and separated by twelve -CAGT- repeats. Another pMir-Reportβ-gal Control vector was used as a no reporter control. We transfected200 ng of the reporter vector into fifty thousand mirPS cells in theabsence or presence of Dox treatment, using a FuGene HD reagent (Roche)following the manufacturer's suggestion. Cell lysates were harvested 48hours after transfection, and the knockdown levels of luciferase werenormalized and shown by ratios of relative luciferase activity (RFA),which was calculated by the level of luciferase activity in Dox-treated(Dox-on) mirPS cells divided by that of untreated (Dox-off) mirPS cells.Mir-434-expressing cells generated by electroporating hHFCs withpTet-On-tTS-miR434-5p were served as a negative control. The result wasshown in FIG. 7B.

Example 16 TRAP Assay

Cells (10⁶) were lysed with a CelLytic-M lysis/extraction reagent(Sigma) supplemented with protease inhibitors, Leupeptin, TLCK, TAME andPMSF, following the manufacturer's suggestion. Lysates were centrifugedat 12,000 rpm for 20 min at 4° C. and the supernatant was recovered.Protein concentrations were measured using an improved SOFTmax proteinassay package on an E-max microplate reader (Molecular Devices, Calif.).Oligonucleotides 5′-AATCCGTCGAGCAGAGTT-3′ (SEQ.ID.NO.24) labeled withinfrared Alexa Fluor 680 dye (TS Primer; Sigma-Genosys) and5′-GTGTAACCCTAACCCTAACCC-3′ (CX primer; 30 μM) (SEQ.ID.NO.25) were usedfor detecting the PCR products. Telomerase inhibitors were directlyadded to the master mix. The optimal results for all tested cell lineswere achieved using 50 ng proteins per reaction. After a 30-minincubation at 30° C., the samples were placed in a thermal cycler for 2min at 94° C., followed by 35 PCR cycles of denaturation at 94° C. for30 sec and synthesis at 57° C. for 30 sec as well as a singlepostsynthesis step at 57° C. for 30 sec. The PCR products were separatedby electrophoresis on a 6% nondenaturing polyacrylamide gel and detectedusing Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor).The result was shown in FIG. 9A.

Example 17 Statistic Analysis

Any change over 75% of signal intensity in the analyses ofimmunostaining, western blotting and northern blotting was considered asa positive result, which in turn was analyzed and presented as mean±SE.Statistical analysis of data was performed by one-way ANOVA. When maineffects were significant, the Dunnett's post-hoc test was used toidentify the groups that differed significantly from the controls. Forpairwise comparison between two treatment groups, the two-tailed studentt test was used. For experiments involving more than two treatmentgroups, ANOVA was performed followed by a post-hoc multiple range test.Probability values of p<0.05 was considered significant. All p valueswere determined from two-tailed tests.

REFERENCES

The following references are hereby incorporated by reference as iffully set forth herein:

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art, andare to be included within the spirit and purview of the invention as setforth in the appended claims. All publications and patents cited hereinare incorporated herein by reference in their entirety for all purposes.

TABLE 1 related to Example 4. Probe Sequence Oct3/4 5′-GCAGTGTGGGTTTCGGGCAC TGCAGGAACA AATTCTCCAG GTTGCCTCTC ACTCGGTTCT CGATACTGGTTCGCTTTCTC TTTCGGGCCT GCACGAGGGT TTCTGCTTTG-3′ (SEQ.ID.NO. 26) Sox25′-TGCTGTAGGT GGGCGAGCCG TTCATGTAGG TCTGCGAGCT GGTCATGGAG TTGTACTGCAGGGCGCTCAC GTCGTAGCGG TGCATGGGCT GCATCTGCGC TGCGCCGTGC-3′ (SEQ.ID.NO.27) Nanog 5′-CGTGTGAGGC ATCTCAGCAG AAGACATTTG CAAGGATGGA TAGTTTTCTTCAGGCCCACA AATCACAGGC ATAGGTGAAG ATTCTTTACA GTCGGATGCT TCAAAGCAAG-3′(SEQ.ID.NO. 28) Lin28 5′-AGGTCCGGTG ACACGGATGG ATTCCAGACC CTTGGCTGACTTCTTAAAGG TGAACTCCAC TGCCTCACCC TCCTTCAAGC TCCGGAACCC TTCCATGTGCAGCTTACTCT-3′ (SEQ.ID.NO. 29) UTF1 5′-CTGCTGGGCC AGCGCGGCCG ACACGCGGCGGTAGGTGGGC AGGGCCTGGC GGCGGTCCAG GAGCAGCGCG CGCCACACGG CCGGTTGCAGCAGCGTCCCC AGCAGCAGCT-3′ (SEQ.ID.NO. 30) Klf4 5′-CTGCTCGACG GCGACGACGAAGAGGAGGCT GACGCTGACG AGGACACGGT GGCGGCCACT GACTCCGGAG GATGGGTCAGCGAATTGGAG AGAATAAAGT CCAGGTCCAG-3′ (SEQ.ID.NO. 31) FUT3 5′-GAGCCCTAGGGGATCCAGTG GCATCGTCTC GGGACACACG CAGGTAGGAG AAGAAACACA CAGCCACCAGCAGCTGAAAT AGCAGTGCGG CCAGACAGCG GCGCCATGGC-3′ (SEQ.ID.NO. 32) RGFP5′-CGAAGGGGTT GCCGTCGCCC TCGCCCTCGC ACTTGAAGTA GTGGCCGTTC ACGGTGCCCTCCATGTACAT CTTGATGCGC ATACTCTCCT TCAGCAGGCC GCTCACCATA-3′ (SEQ.ID.NO.33) B-actin 5′-AATGTCACGC ACGATTTCCC GCTCGGCCGT GGTGGTGAAG CTGTAGCCGCGCTCGGTGAG GATCTTCATG AGGTAGTCAG TCAGGTCCCG GCCAGCCAGG TCCAGAGCGA-3′(SEQ.ID.NO. 34)

1. A method for using a recombinant nucleic acid composition to inducespecific gene silencing effects in a targeted cell, comprising the stepsof: (a) providing the recombinant nucleic acid composition capable ofbeing delivered, transcribed and processed into at least a genesilencing effector interfering a plurality of cellular genes targeted bymir-302; and (b) treating a cell substrate containing the targeted celland expressing mir-302-targeted cell cycle regulators and oncogenes withsaid recombinant nucleic acid composition.
 2. The method as defined inclaim 1, wherein said targeted cell includes a human cell categorized asone of tumor types found in teratoma.
 3. The method as defined in claim1, wherein said targeted cell includes a human cell derived fromcancerous tissues.
 4. The method as defined in claim 1, wherein saidrecombinant nucleic acid composition includes an expression-competentvector.
 5. The method as defined in claim 1, wherein said recombinantnucleic acid composition includes a drug-inducible gene expressionpromoter.
 6. The method as defined in claim 5, wherein saiddrug-inducible gene expression promoter is controlled by a tetracyclinederivative or equivalent.
 7. The method as defined in claim 1, whereinsaid recombinant nucleic acid composition includes a constitutive geneexpression promoter.
 8. The method as defined in claim 7, wherein saidconstitutive gene expression promoter is driven by a type-II RNApolymerase or equivalent.
 9. The method as defined in claim 7, whereinsaid constitutive gene expression promoter is a viral promoter.
 10. Themethod as defined in claim 1, wherein said cell substrate expressing aplurality of cellular genes targeted by mir-302.
 11. The method asdefined in claim 1, wherein said treating cell substrate is under acondition that said cellular genes targeted by mir-302 are suppressed.12. The method as defined in claim 1, wherein said recombinant nucleicacid composition includes a 5′-donor splice site, an intronic insertsite, a branch point motif, a poly-pyrimidine tract, and a 3′-acceptorsplice site.
 13. The method as defined in claim 12, wherein saidintronic insert site includes said gene silencing effector homologous tomir-302.
 14. The method as defined in claim 1, wherein said recombinantnucleic acid composition further includes a plurality of exons.
 15. Themethod as defined in claim 1, wherein said gene silencing effectorcontains a sequence homologous to either a SEQ.ID.NO.1 or a SEQ.ID.NO.2sequence.
 16. The method as defined in claim 1, wherein said genesilencing effector is homology or complementarity, or both, to aSEQ.ID.NO.3 sequence.
 17. The method as defined in claim 1, wherein saidgene silencing effector is formed by ligation linkage of hybrids of aSEQ.ID.NO.9, a SEQ.ID.NO.10, a SEQ.ID.NO.11, a SEQ.ID.NO.12, aSEQ.ID.NO.13, a SEQ.ID.NO.14, a SEQ.ID.NO.15, a SEQ.ID.NO.16 sequence,and a combination thereof.
 18. The method as defined in claim 1, whereinsaid gene silencing effector is a recombinant nucleic acid sequenceformed by the hybrid of a SEQ.ID.NO.17 and a SEQ.ID.NO.18 sequence. 19.The method as defined in claim 1, wherein said recombinant nucleic acidcomposition is delivered into said cell substrate by a gene deliverymethod.
 20. The method as defined in claim 1, wherein saidmir-302-targeted cell cycle regulators and oncogenes are selected fromthe group consisting of CDK2, CDK4, CDK6, cyclin D, BMI-1, and acombination thereof.
 21. The method as defined in claim 1, wherein saidspecific gene silencing effects include at least one of cell cycleattenuation, G0/G1-checkpoint arrest, tumor suppression,anti-tumorigenecity, and cancer cell apoptosis.
 22. The method asdefined in claim 1, wherein said specific gene silencing effects resultfrom RNA interference.
 23. The method as defined in claim 1, whereinsaid recombinant nucleic acid composition is made by a geneticengineering method.